Modular targeted liposomal delivery system

ABSTRACT

A liposome including a fusogenic liposome, a linking moiety and a targeting moiety. The fusogenic liposome is a lipid bilayer encapsulating contents. The linking moiety is electrostatically bound to the lipid bilayer, and the targeting moiety is covalently bound to the linking moiety. The liposome may also include a stabilizing moiety interposed between the linking and targeting moieties, and covalently bound to both. Alternatively, the stabilizing and targeting moieties may be covalently bound to separate linking moieties, the linking moieties being electrostatically bound to the lipid bilayer.

FIELD OF THE INVENTION

The invention relates to a targeted liposome capable of efficientintracellular delivery of one or more bioactive agents, a pharmaceuticalcomposition including the targeted liposome, and a method of deliveringthe contents of a liposome to an in vivo or in vitro target site such asa tumor cell, site of inflammation or infection.

BACKGROUND OF THE INVENTION

Pharmaceutical usefulness of bioactive agents depends upon the abilityto position therapeutically-effective quantities of intact agent at thetarget site in the patient. Delivering intact bioactive agents to targetsites can be difficult: In vivo degradation of bioactive agents canoccur, as well as absorption/retention of the agent by non-targetedsystems. Even if pharmaceutically effective amounts of intact agent canbe delivered to the vicinity of the target site, accessing thefunctional location of the site for the bioactive agent can bechallenging, particularly if that location is intracellular: Forexample, certain polar compounds and many large molecules can not entercells at all because of their inability to cross target cell membranes.In addition, dilution of the bioactive agent by non-specific binding tonon-target sites reduces the amount of bioactive agent available to thetarget site.

Yet another challenge in the therapeutic delivery of drugs or bioactiveagents is limiting the toxicities often associated with therapeuticallyeffective concentrations of drugs or bioactive agents. Delivery to aspecific target site can also reduce some toxicity normally associatedwith the administration of a drug or agent. Even when a drug orbioactive agent has no toxicity associated with it, the “loss” of agentthrough degradation, removal by non-target organs and other deliveryfailures can significantly and prohibitively increase the cost of thetherapy or decrease the efficacy.

One method used to deliver bioactive agents or drugs to tissues andcells is to encapsulate the bioactive agents or drugs in liposomes.Often, an added advantage to this type of formulation is the reductionof the toxicity associated with certain drugs or bioactive agents. Theintracellular targeting possibilities that liposomes provide areespecially intriguing. While certain cells are known to engulfliposomes, delivery of most liposomes to a target site is not sufficientto deliver the encapsulated contents to the interior of the cell.Fusogenic liposomes are known that allow the liposome's bilayer to fusewith the cell membrane and thus, deliver the encapsulated bioactiveagents or drugs to the cell. However, often these fusogenic liposomeslack stability when incubated in serum. In addition, most fusogenicliposomes have not heretofore been able to be targeted to the specificsite where the bioactive agent or drug is required. Efficient liposomaldelivery to cells in vivo requires specific targeting and substantialprotection from the extracellular environment, particularly serumproteins. Unfortunately, most known targeting and protecting strategiesalso generate large steric barriers on the surface of the liposomes thatlimit or prohibit fusogenic delivery of the liposomal contents into theinterior of the cell. In addition, known targeting and protectingstrategies attach targeting/protecting molecules to the liposome throughhydrophobic bonding to the liposome's lipid bilayer. These hydrophobicbonds usually inhibit the function of fusogenic membranes. When thehydrophobic moiety is designed to allow dissociation of thetargeting/protecting moiety, its dissociation does not occurspecifically at the target site.

SUMMARY OF THE INVENTION

Briefly, the invention is a modular liposomal targeting and deliverysystem comprising a fusogenic liposome, a linking moiety and a targetingmoiety. In an alternate embodiment, the system may comprise astabilizing moiety, either instead of or in addition to the targetingmoiety. The fusogenic liposome comprises a lipid bilayer encapsulatingcontents to be delivered to a target site such as a tumor, a site ofinflammation or infection and/or to the cytoplasm of a cell. Theliposomes may contain one or more bioactive agents or drugs or maycontain a combination of bioactive agents and drugs. In one embodiment,the bioactive agent is a nucleic acid, preferably DNA. The targetingand/or the stabilizing moiety is covalently bound to one or more linkingmoieties, and the linking moiety electrostatically binds at neutral pHto the lipid bilayer of the fusogenic liposome. The linking moiety isselected from the group consisting of polylysine, protamine,polyethyleneimine, polyarginine, polyacrylate, a spermine derivative,cytochrome c, an annexin, heparin sulfate, an aminodextran,polyaspartate, polyglutamate, a polysialic acid, and/orpoly(2-ethylacrylic acid). The targeting moiety is a molecule that willbind selectively to the surface of targeted cells. For instance, thetargeting molecule may be a ligand that binds to the cell surfacereceptor found on a particular cell type or expressed at a higherfrequency on target cells than on other cells. The targeting moiety isselected from the group consisting of a vitamin, transferrin, anantibody or fragment thereof, sialyl Lewis X antigen, hyaluronic acid,mannose derivatives, glucose derivatives, cell specific lectins,galaptin, galectin, lactosylceramide, a steroid derivative, an RGDsequence, EGF, EGF-binding peptide, urokinase receptor binding peptide,a thrombospondin-derived peptide, an albumin derivative and/or amolecule derived from combinatorial chemistry. The targeting moietyenhances the ability of the liposome to bind to a targeted cell and todeliver the liposomal contents to the target site. While not beinglimited to the following explanation of the mechanism by which thepresent invention delivers the liposomal contents to the cellularinterior, it has been theorized that after the liposome binds to a cell,an endocytosis pathway may be provoked, wherein the liposome is engulfedand sequestered in an endosomal compartment within the cell. The low pH(relative to the extra-cellular plasma) within the endosomal compartmentweakens the electrostatic bond between the linking moiety and theliposome, wherein at least a portion of the linking moiety (andtargeting moiety covalently bound thereto) dissociates at leasttemporarily from the liposome to expose the fusogenic lipid bilayer andenable fusion of the liposomal membrane and endosomal membrane. Releaseof the liposomal contents into the cytoplasm of the cell results fromthe fusion. Even if the endocytosis pathway is not initiated when theliposome binds to the cell, a low pH immediately surrounding the cell(relative to the plasma pH beyond the immediate vicinity of the cell)could cause at least temporary dissociation of the linking moiety fromthe liposome as described above, thereby enabling release of theliposomal contents into the cell cytoplasm.

Preferably, the invention also comprises a stabilizing moiety that isindirectly attached to the lipid bilayer of the fusogenic liposomethrough a linking moiety. The stabilizing moiety is covalently bound tothe linking moiety, which linking moiety is electrostatically bound orcan be electrostatically bound to the lipid bilayer at or near neutralpH, i.e., the pH of normal serum. A targeting moiety may also becovalently bound to the stabilizing moiety. Preferably, the stabilizingmoiety is selected from the group consisting of polyethylene glycol,polyvinylpyrolidone, a dextran, a polyamino acid, methyl-polyoxazoline,polyglycerol, poly(acryloyl morpholine), and/or polyacrylamide.

In one embodiment, the invention is a composition comprising a fusogenicliposome comprising a lipid bilayer encapsulating contents; and alinking moiety electrostatically bound to said lipid bilayer; wherein atargeting moiety is covalently bound to said linking moiety.

The invention also encompasses a method of introducing a bioactive agentinto a cell, comprising preparing a fusogenic liposome having a lipidbilayer which encapsulates a bioactive agent, electrostatically linkinga targeting moiety to said fusogenic liposome to form a targetedliposome, contacting the targeted liposome with a cell such that thetargeting moiety is released from at least a portion of the lipidbilayer of the liposome to expose a portion of the lipid bilayer, andfusing the exposed lipid bilayer portion with a cell membrane such thatthe bioactive agent is released into the cell. Preferably, the methodalso includes electrostatically binding a stabilizing agent to the lipidbilayer of the fusogenic liposome.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts transfection of ovarian cancer cells by charge reversalliposomes and its enhancement by the use of a targeting/stabilizingmodule.

FIG. 2 depicts the quantitation of enhanced transfection efficiency forcharge reversal liposomes when targeted by modules with transferrin orfolate.

FIG. 3 demonstrates that enhanced transfection efficiency of fusogenicANacylPE liposomes by a folate targeting module is dependent on folate.

FIG. 4 demonstrates that folate targeting modules electrostaticallylinked to the liposomes enhance transfection more than folate conjugatedto lipids.

FIG. 5 depicts the maintenance of liposomal integrity when an antibodyconjugate is electrostatically bound to the liposome surface.

FIG. 6 demonstrates enhanced liposomal uptake mediated by an antibodyconjugate electrostatically bound to the liposome surface.

FIG. 7 shows enhanced transfection as a result of increasing amounts ofan antibody conjugate electrostatically bound to the surface ofliposomes containing plasmid DNA.

FIG. 8 depicts enhanced green fluorescent protein expression as a resultof antibody targeting.

FIG. 9 depicts the binding of transferrin-targeted liposomes of thepresent invention to OVCAR-3 cells in the presence and absence of serum.

FIG. 10 depicts the effect of polylysine-transferrin congugates onliposome binding and transfection.

FIG. 11 is a fluorescent photomicrograph depicting the effect of pKT onliposome binding to cells and transfection.

FIG. 12 demonstrates the inhibition of transfection mediated bypKT-targeted liposomes by anti-transferrin antibodies.

FIG. 13 depicts the transfection efficiency of conjugates compared tofree polylysine.

FIG. 14 demonstrates the binding of fluorescent liposomes to OVCAR-3cells derived from the ascites of a mouse xenograft with and without atargeting agent (pKT).

FIG. 15 demonstrates the beta-galactosidase assay for in vivotransfection.

FIG. 16 demonstrates the beta-galactosidase assay for in vivotransfection.

DETAILED DESCRIPTION OF THE INVENTION

Delivery of liposomally encapsulated drugs or nucleic acids to specificcells via membrane fusion is a highly desirable goal because ofprotection of the cargo from degradation, potential for targeting anddecrease of drug toxicity. Furthermore, it is advantageous to protectand/or stabilize the liposome itself until it reaches its desired siteof delivery, because the liposome can be degraded in vivo. However, manytargeting molecules such as antibodies and most protecting orstabilizing moieties such as polyethylene glycol (PEG) stericallyinhibit the interaction between the liposomal membrane and the cellmembrane, even though the liposome has bound to the cell surface.Because of this steric hindrance, it is generally not possible forfusogenic liposomes to efficiently deliver contents to the cytoplasm ofthe cell. This invention provides a targeted liposome, or targeted andstabilized liposome, where at least a portion of thetargeting/stabilizing moieties are at least temporarily removable at thetarget site. The invention involves non-covalent adherence of atargeting moiety, and preferably a stabilizing moiety, to the surface ofa liposome by electrostatic interactions, preferably multivalentelectrostatic interactions. Such interactions can be quite strong if thevalency of interaction is high enough. However, the reduced pH (relativeto the extra-cellular plasma beyond the immediate vicinity of the cell)at certain in vivo sites such as the site of infection, inflammation orwithin certain cellular organelles or compartments such as endosomes,loosens or, in certain cases, reverses the interaction between theadherent moiety and the liposome. Once a fusogenic liposome, somodified, binds to the targeted cell type and encounters the reduced pHenvironment, the targeting/stabilizing moiety may be completely orpartially removed if at least one of the electrostatically interactingspecies, i.e., membrane lipids or targeting/stabilizing agent is atleast partially neutralized by the low pH environment in combinationwith other ions. Furthermore, a portion of the electrostatically boundtargeting/stabilizing agent may exchange onto the cellular membranefollowing binding of the liposome and internalization into an endosome,also eliminating steric interference to fusion. For example, afusogenic, anionic liposome can be protected by a polylysine conjugatewith PEG and further targeted by a linked molecule, such as folate,transferrin or an antibody. If the anionic lipid is at least partiallyneutralized at lower pH, the targeting/stabilizing agent may completelyor partially dissociate allowing exposure of the bare fusogenicliposomal membrane and subsequent fusion or partial fusion of theliposome with the endosomal membrane. This releases the liposomalcontents into the cytoplasm. The contents may include anytherapeutically relevant molecule(s).

A second advantage of this method of targeting and stabilizing theliposome is that a single type of liposome can be produced to which anynumber of targeting/stabilizing agents can be subsequently boundelectrostatically. This embodiment merely requires mixing a fusogenicliposome, encapsulating a relavant drug or bioactive agent, with alinking moiety covalently bound to a targeting moiety that will bind tothe specific targeted site. Thus, the system is modular. Selectedtargeting agents covalently linked to linking moieties that will bindelectrostatically to the lipid bilayer can be prepared. Fusogenicliposomes encapsulating different drug or bioactive agents can beindependently prepared. Then, depending on the target site and thetherapeutic agent(s) one desires to administer, the appropriatecombination of targeting agent and liposome encapsulating bioactiveagent or drug can be mixed to provide a specific therapeutic agent thatis targeted to the specific cellular site. This will allowpatient-specific targeting with a single type of liposomal preparationthat can be modified by an array of targeting agents.

The basic elements of the invention are a fusogenic liposome, a linkingmoiety and a targeting moiety. The fusogenic liposome comprises a lipidbilayer encapsulating contents to be delivered to the cytoplasm of acell, the contents preferably being a bioactive agent, and morepreferably condensed DNA. The targeting moiety is positioned exteriorlyof the liposome, and is covalently bound to the linking moiety. Thelinking moiety is electrostatically bound to the lipid bilayer of thefusogenic liposome, thus indirectly linking the targeting moietyelectrostatically to the lipid bilayer. Preferably, the invention alsocomprises a stabilizing moiety which, like the targeting moiety, isindirectly electrostatically bound to the lipid bilayer through alinking moiety. Specifically, the stabilizing moiety is covalently boundto the linking moiety, and the linking moiety is electrostatically boundto the lipid bilayer of the fusogenic lipid. A targeting moiety may becovalently bound to the stabilizing moiety. These individual elements ofthe invention will now be described in detail.

“Liposomes” are self-assembling structures comprising one or more lipidbilayers, each of which comprises two monolayers containing amphipathiclipid molecules oppositely oriented. Amphipathic lipids comprise a polar(hydrophilic) headgroup region covalently linked to one or two non-polar(hydrophobic) acyl chains. Energetically unfavorable contacts betweenthe hydrophobic acyl chains and the surrounding aqueous medium inducethe amphipathic lipid molecules to arrange themselves such that theirpolar headgroups are oriented towards the bilayer's surface, while theacyl chains reorient towards the interior of the bilayer. Anenergetically stable structure is thus formed in which the acyl chainsare effectively shielded from coming into contact with the aqueousenvironment. A “fusogenic” liposome as defined herein is a liposome thatis capable of interacting with a cell membrane in a way that permits thecontents of the liposome to enter the cytoplasm of the cell. Withoutlimitation, this interaction can take the form of complete or partialfusion of the membranes, or adjacent, contemporaneous and localizeddisruptions of the cellular and liposomal membranes that allows passageof the liposome contents into the cytoplasm of the cell.

Liposomes (see, e.g., Cullis et al., 1987; New, 1995) can have a singlelipid bilayer (unilamellar liposomes, “ULVs”), or multiple lipidbilayers (multilamellar liposomes, “MLVs” or “SPLVs”). Each bilayersurrounds, or encapsulates, an aqueous compartment. Given thisencapsulation of aqueous volume within a protective barrier of lipidmolecules, liposomes are able to sequester encapsulated molecules, e.g.,nucleic acids, away from the degrading effects of factors, e.g.,nuclease enzymes, present in the external environment.

Liposomes can have a variety of sizes, e.g., an average diameter as lowas 25 nm or as high as 10,000 nm or more. Size is affected by a numberof factors, e.g., lipid composition and method of preparation, wellwithin the purview of ordinarily skilled artisans to determine andaccount for, and is determined by a number of techniques, such asquasi-elastic light scattering, also within the artisans' purview.

Various methodologies, also well within the purview of ordinarilyskilled artisans, such as sonication, homogenization, French Pressapplication and milling can be used to prepare liposomes of a smallersize from larger liposomes. Extrusion (see, e.g., U.S. Pat. No.5,008,050) can be used to size reduce liposomes, that is to produceliposomes having a predetermined mean size by forcing the liposomes,under pressure, through filter pores of a defined, selected size.Tangential flow filtration (WO89/008846), can also be used to regularizethe size of liposomes, that is, to produce a population of liposomeshaving less size heterogeneity, and a more homogeneous, defined sizedistribution. The contents of these documents are incorporated herein byreference.

Liposomes of this invention can be unilamellar, or oligolamellar, andcan have a size equal to that of liposomes produced by any of themethods set forth hereinabove. However, in preferred embodiments of thisinvention, the liposomes are unilamellar liposomes having number averagesizes of about 50-500 nm.

Liposomes are composed of a variety of lipids, both amphipathic andnonamphipathic, obtained from a variety of sources, both natural andsynthetic. Suitable liposomal lipids include, without limitation,phospholipids such as phosphatidylcholines (“PC's”),phosphatidylethanolamines (“PE's”), phosphatidylserines (“PS's”),phosphatidylglycerols (“PG's”), phosphatidylinositols (“PI's”) andphosphatidic acids (“PA's”). Such phospholipids generally have two acylchains, these being either both saturated, both unsaturated or onesaturated and one unsaturated; said chains include, without limitation:myristate, palmitate, stearate, oleate, linoleate, linolenate,arachidate, arachidonate, behenate and lignocerate chains.

Phospholipids can also be derivatized, by the attachment thereto of asuitable reactive group. Such a group is generally an amino group, andhence, derivatized phospholipids are typicallyphosphatidylethanolamines. The different moieties suited to attachmentto PE's include, without limitation: acyl chains (WO98/16199), usefulfor enhancing the fusability of liposomes to biological membranes;peptides (WO 98116240), useful for destabilizing liposomes in thevicinity of target cells; biotin and maleimido moieties (U.S. Pat. Nos.5,059,421 and 5,399,331, respectively), useful for linking targetingmoieties such as antibodies to liposomes; and, various molecules such asgangliosides, polyalkylethers, polyethylene glycols and organicdicarboxylic acids (see, e.g., U.S. Pat. Nos. 5,013,556, 4,920,016 and4,837,028). The contents of the above-cited documents are incorporatedherein by reference.

Accordingly, in the most preferred embodiments of this invention, theliposomes comprise a derivatized phospholipid, adapted so as to enhancedelivery of their contents. The liposomes may also, but are not requiredto, comprise additional lipids as well, said additional lipids beingincorporated into the liposomes for a number of reasons apparent toartisans of ordinary skill in the field of liposomology. Such reasonsinclude, without limitation, stabilizing or targeting the liposomes, aswell as further altering the liposomes' pharmacokinetic behavior.Suitable additional lipids include any of those lipids commonlyrecognized as suitable for incorporation in liposomes, including,without limitation, phospholipids, glycolipids and sterols.

Preferably, liposomes of this invention have a lipid component whichcomprises a derivatized phospholipid and an additional lipid. Suitableliposomes and the methods of preparing them are described in U.S. patentapplication Ser. No. 08/951,056, incorporated herein in its entirety byreference.

Preferably, the derivatized phospholipid is an N-acylated PE. Such NAPEsare useful in preparing fusogenic liposomes and are preferred forpreparing liposomes comprising the drug or bioactive agent complexes ofthe present invention.

NAPE-induced bilayer destabilization induces the bilayers to fuse tobiological membranes in the vicinity and hence, enhances the bilayers'fusogenicity (Shangguan et al., 1998). Enhanced fusogenicity, in turn,can be used to deliver encapsulated bioactive agents, such as nucleicacids or other agents that can not cross the cell membrane, to cells, bycombining the cells with the liposomes under conditions, e.g., thepresence of appropriate concentrations such as Ca^(t)+ and Mg²⁺.Liposome-cell contact results in release of the liposome-encapsulatedbioactive agents local to the cells, and/or directly into the cells'cytoplasm as a result of fusion between liposome and cell membranes.Such delivery is either in vivo or in vitro.

An alternative preferable formulation of liposomes for modular targetingare referred to below (example 1) as “charge reversal” liposomes. Thecomposition of such liposomes also allows electrostatically boundtargeting/stabilizing groups. Such liposomes reverse charge at low pH,dissociating the targeting/stabilizing conjugates and attaining apositive charge to enhance interaction with the cellular membrane.

The liposomal lipid can also comprise a “headgroup-modified lipid,”i.e., a lipid having a polar group derivatized by the attachment theretoof a moiety capable of inhibiting the binding of serum proteins to aliposome incorporating the lipid. Incorporation of headgroup-modifiedlipids into liposomes thus alters their pharmacokinetic behavior, suchthat the liposomes remain in the circulation of an animal for a longerperiod of time then would otherwise be the case (see, e.g., Blume etal., 1993; Gabizon et al., 1993; Park et al., 1992; Woodle et al., U.S.Pat. No. 5,013,556; Allen et al., U.S. Pat. Nos. 4,837,028 and4,920,016; the contents of these documents being incorporated herein byreference).

Nucleic acids that may be encapsulated in the liposome are DNA,including genomic DNA, plasmid DNA and cDNA, or RNA; preferably, theencapsulated nucleic acid is DNA, more preferably, closed (circular)plasmid DNA. “Encapsulated” or “containing” as used herein with regardto the contents of the liposome describes materials that are within theinterior aqueous volume of the liposome, intercalated in the lipidbilayer of the liposome, or partly intercalated in the lipid bilayer ofthe liposome.

Liposomes of the invention can contain one or more bioactive agents.Bioactive agents which may be associated with liposomes include, but arenot limited to: antiviral agents such as acyclovir, zidovudine and theinterferons; antibacterial agents such as aminoglycosides,cephalosporins and tetracyclines; antifungal agents such as polyeneantibiotics, imidazoles and triazoles; antimetabolic agents such asfolic acid, and purine and pyrimidine analogs; antineoplastic agentssuch as the anthracycline antibiotics and plant alkaloids; sterols suchas cholesterol; carbohydrates, e.g., sugars and starches; amino acids,peptides, proteins such as cell receptor proteins, immunoglobulins,enzymes, hormones, neurotransmitters and glycoproteins; dyes;radiolabels such as radioisotopes and radioisotope-labeled compounds;radiopaque compounds; fluorescent compounds; mydriatic compounds;bronchodilators; local anesthetics; and the like.

Liposomal bioactive agent formulations can enhance the therapeutic indexof the bioactive agent, for example by buffering the agent's toxicity.Liposomes can also reduce the rate at which a bioactive agent is clearedfrom the circulation of animals. Accordingly, liposomal formulation ofbioactive agents can mean that less of the agent need be administered toachieve the desired effect.

The liposome of this invention may be dehydrated, stored and thenreconstituted such that a substantial portion of their internal contentsare retained. Liposomal dehydration generally requires use of ahydrophilic drying protectant such as a disaccharide sugar at both theinside and outside surfaces of the liposomes' bilayers (see U.S. Pat.No. 4,880,635, the contents of which are incorporated herein byreference). This hydrophilic compound is generally believed to preventthe rearrangement of the lipids in liposomes, so that their size andcontents are maintained during the drying procedure, and throughsubsequent rehydration. Appropriate qualities for such dryingprotectants are that they be strong hydrogen bond acceptors, and possessstereochemical features that preserve the intermolecular spacing of theliposome bilayer components. Alternatively, the drying protectant can beomitted if the liposome preparation is not frozen prior to dehydration,and sufficient water remains in the preparation subsequent todehydration.

Also provided herein is a pharmaceutical composition comprising apharmaceutically acceptable carrier and the liposome of this invention.Said composition is useful, for example, in the delivery of nucleicacids to the cells of an animal. “Pharmaceutically acceptable carriers”as used herein are those media generally acceptable for use inconnection with the administration of lipids and liposomes, includingliposomal bioactive agent formulations, to animals, including humans.Pharmaceutically acceptable carriers are generally formulated accordingto a number of factors well within the purview of the ordinarily skilledartisan to determine and account for, including without limitation: theparticular liposomal bioactive agent used, its concentration, stabilityand intended bioavailability; the disease, disorder or condition beingtreated with the liposomal composition; the subject, its age, size andgeneral condition; and the composition's intended route ofadministration, e.g., nasal, oral, ophthalmic, topical, transdermal,vaginal, subcutaneous, intramammary, intraperitoneal, intravenous, orintramuscular (see, for example, Naim (1985), the contents of which areincorporated herein by reference). Typical pharmaceutically acceptablecarriers used in parenteral bioactive agent administration include, forexample, D5W, an aqueous solution containing 5% weight by volume ofdextrose, and physiological saline. Pharmaceutically acceptable carrierscan contain additional ingredients, for example those which enhance thestability of the active ingredients included, such as preservatives andanti-oxidants.

Lipids useful in the practice of this invention are, as describedhereinabove, those lipids recognized as suitable for incorporation inliposomes, either on their own or in connection with additional lipids;these include, phospholipids, glycolipids, sterols and theirderivatives. Organic solvents used in this method are any of the varietyof solvents useful in dissolving lipids during the course of liposomepreparation; these include, without limitation, methanol, ethanol,dimethylsulfoxide, chloroform, and mixtures thereof Preferably, theorganic solvent is chloroform or methylene chloride.

Still further provided herein is a method of transfecting the cells ofan animal with a targeted fusogenic liposome comprising one or morebioactive agents such as, but not limited to, a nucleic acid. The methodcomprises the steps of preparing a fusogenic liposome encapsulating abioactive agent; preparing a targeting moiety covalently bound to alinking agent said linking agent being capable of electrostaticallybinding to said fusogenic liposome at normal physiological pH, mixingthe loaded fusogenic liposome with the targeting conjugate andcontacting the cells with the composition comprised of the loadedfusogenic liposome electrostatically bound to the targeting agent. Suchcontact is either in vitro, in which case, a composition comprising theliposome is added to the culture medium surrounding the cells, or invivo, in which case the liposome is administered in a pharmaceuticalcomposition also comprising a pharmaceutically acceptable carrier, andis administered to the animal by any of the standard means ofadministering such compositions to animals.

The targeting moiety of the invention can be any chemical compositionthat favors the positioning of a liposome to a specific site or sites.More than one targeting moiety may be utilized on a single liposome.Preferably, the targeting moiety is selected from the group consistingof a vitamin such as folate; transferrin; an antibody such as OVB-3,anti-CAl₂5, anti-CEA, and others; sialyl Lewis X antigen, hyaluronicacid, mannose derivatives, glucose derivatives, cell specific lectins,galaptin, galectin, lactosylceramide, a steroid derivative, an RGDsequence, a ligand for a cell surface receptor such as epidermal growthfactor (EGF), EGF-binding peptide, urokinase receptor binding peptide, athrombospondin-derived peptide, an albumin derivative and/or acombinatorial molecule directed against various cells.

The linking moiety can be any chemical composition that is capable ofsimultaneously binding to the lipid bilayer of a liposomeelectrostatically and binding to a targeting or stabilizing moietycovalently, such that the electrostatic bond can be so weakened inreduced pH environment (relative to the extra-cellular plasma not in theimmediate vicinity of the cell) so that at least some of the linkingmoiety at least temporarily dissociates from the liposome, therebyexposing at least a portion of the outer lipid bilayer for at least someperiod of time. The linking moiety is preferably a small molecule thatdoes not hinder the interaction between the targeting moiety andtargeted cell. More than one linking moiety can be used on a singleliposome, and the linking moieties used to bind the targeting andstabilizing moieties may be the same or different. Preferably, thelinking moiety is selected from the group consisting of polylysine,protamine, polyethyleneimine, polyarginine, polyacrylate, a sperminederivative, cytochrome c, an annexin, heparin sulfate, an aminodextran,polyaspartate, polyglutamate, a polysialic acid, and/orpoly(2-ethylacrylic acid).

The stabilizing moiety of the invention is any chemical composition thatinhibits or prevents a liposome from fusing with other liposomes ornon-target cells, and/or protects the lipid bilayer of the liposome fromthe disruptive, degrading or interfering action of detrimental compounds(e.g. serum proteins). More than one stabilizing moiety may be used on asingle liposome. Preferably, the stabilizing moiety is selected from thegroup consisting of polyethylene glycol, polyvinylpyrolidone, a dextran,a polyamino acid, methyl-polyoxazoline, polyglycerol, poly(acryloylmorpholine), and/or polyacrylamide.

Preferred embodiments of the invention will now be described in theExamples below. The Examples are intended to be illustrative of theinvention and are not intended to limit the scope of the inventiondefined in the appended claims.

EXAMPLE 1

Formulations

Preparation of Liposomes for Delivery of Nucleic Acids:

Preferably, for the delivery of nucleic acids to cells, liposomes areprepared according to the method described in U.S. ProvisionalApplication Ser. No. 60/122,365 entitled “Encapsulation of BioactiveComplexes in Liposomes”, filed on Mar. 2.1999. One embodiment isdescribed below.

Plasmid Purification: Two plasmids were used in this study: thepZeoSVLacZ plasmid which is 6.5 kb, and expresses the IacZ gene forβ-galactosidase in mammalian cells from the SV40 earlyenhancer-promoter, allowing selection in mammalian cells and E. coliusing the antibiotic zeocin; and, the pEGFP-Cl plasmid, which is 4.7 kband expresses enhanced green fluorescent protein (EGFP) from a humancytomegalovirus immediate early promoter, allowing selection in E. coilusing kanamycin, and in mammalian cells using G418. Plasmids werepurified from E. Coli (Baumann and Bloomfield, 1995; or by Qiagen kitper manufacturer's instructions (Qiagen, Valencia, Calif.))—the finalratio of O.D. at 260 nm to O.D. at 280 nm was greater than 1.9 for allpreparations; agarose gel electrophoresis indicated DNA in the expectedsize range.

Preparation of Liposomal-DNA Formulations (Fusogenic N-Acyl-PELiposomes: Two Step Emulsion Method):

Samples were prepared by diluting 200 Ag of DNA into 125 Al of low saltbuffer (LSB; 10 mM TrisHCl, 1 mM NaCl, pH 7.0, all LSB used in thesepreparations also contained 200 mM sucrose), and then combining theresulting suspension with 1 ml CHCl₃ containing 30 gmole of 70:30 molarratio of N-Cl₂ DOPE and DOPC, in a 13×100 Pyrex tube while vortexing.The sample was immediately sonicated for 12 seconds in a bath sonicator(Laboratory Supplies Co. Hicksville, N.Y.) under maximum power, to forman emulsion with plasmid DNA first. Subsequently, a 125 μ aliquot of LSBcontaining spermine (preferably 16 to 40 millimolar) was added to thisemulsion with vortexing and sonication.

Resulting emulsions were placed, within a few minutes, in a flask on aRotovap (Buchi Laboratoriums-Technik AG, Switzerland). Organic solventwas removed while rotating the flask at its maximum rate, while thevacuum was modulated with a pin valve. Initially a vacuum ofapproximately 600-650 mm was established, this being subsequentlyincreased, as rapidly as possible without excessive bubbling, until themaximum vacuum was reached (approximately 730 mm); the flask was thenevacuated for another 25 minutes. The film left on the flask wasresuspended in 1 ml of 300 mM sucrose in LSB, and the sample wasextruded five times through 0.4 lam polycarbonate membrane filters(Poretics, Livemore, Calif.). The sample was then dialyzed againstHank's balanced salt buffer (HBSS) without Ca²⁺/Mg²⁺, overnight at 4° C.

After preparation of the nucleic acid-containing liposomes describedabove, the free plasmid DNA was separated from liposome-encapsulated DNAby centrifugation. The liposomes were pelleted and washed prior toforming the targeting complex. The preparations may be scaled-up toproduce larger amounts of product by methods known in the art.

Preparation of Charge Reversal Liposomal DNA Formulations:

In another embodiment, liposomes, heretofore referred to as chargereversal liposomes, were made by first mixing the lipids in chloroform(DOPC:POPE:cholesterol:cholesteryl hemisuccinate:DOPAP:oleyl actetate,12:50:2.5:12.5:10.5:10.5). Next, plasmids were condensed with spermineusing the two-step lipid emulsion protocol previously described in theembodiment above. One difference is that the final sucrose concentrationin the DNA solution to be encapsulated was 300 mM. Solvent was removedby either rotary evaporation or sparging with nitrogen. The liposomeswere suspended from the resulting paste in 300 mM sucrose, extrudedthrough 0.4 μm filters, then dialyzed against PBS or Hanks balanced saltsolution (HBSS). Unencapsulated plasmid was removed by centrifuging thesample at 10,000 g. The liposome/plasmid pellet was washed, respun andresuspended in PBS or HBSS. The size distribution of the liposomes wasdetermined by light scattering using a NICOMP model 370 submicronparticle sizer (NICOMP, Santa Barbara, Calif.). The amount of plasmidpresent in the liposomal pellet was quantitated using a PicoGreenfluorescent assay (Molecular Probes, Inc., Eugene, Oreg.) as describedin Example 3 “Assays”.

Liposomes of this composition were characterized by transmissionelectron microscopy of a liposome/plasmid preparation withpolylysine-anti-OVB3 ligand attached. The sample was placed on a carboncoated EM grid and negatively stained with 1% uranyl acetate. Liposomesof a heterogenous size were observed with varying amounts of stainpenetration. The final liposome/plasmid preparations gave a Gaussiansize distribution by light scattering analysis. The number weighted meandiameter of 7 different preparations was 140 nm±20 nm (standarddeviation of the mean for any single preparation ranged between 40-55%).Plasmid content in the washed liposome/plasmid samples ranged from1.25-2.45 μg DNA/gmol lipid, with a mean of 1.6 μg DNA/μmol lipid. Thisplasmid fraction was protected from digestion with DNAse I suggestingeither encapsulation or very tight association with the liposomes.

EXAMPLE 2

Synthesis of Targetingistabilizing Moiety—Linker Conjugates

Targeting/stabilizing modules were synthesized to use with liposomesprepared as in example 1. Targeting/stabilizing moieties may becovalently coupled to a linker for electrostatic interaction, such aspolylysine (pK), by any method known in the art. Examples of processesfor coupling polylysine to targeting agents such as folic acid,transferrin and antibodies are provided herein for exemplary purposesonly. Methods for coupling a linking agent such as polylysine to astablizing agent such as PEG or to a stabilizing agent and a targetingmoiety are also provided herein.

a. Preparation of Folic acid, PEG and Polylysine Without Glutaric Acid(FA-NH-PEG-CO-PL or FPK)

Preparation of FAA

Folic acid (50 mg, 0.11 mmole) was dissolved in 6 ml of DMF:PY (5:1;v/v). To this solution dicylohexylcarbodiimide (DCC, 140 mg, 0.68mmoles) was added and the reaction mixture was stirred at roomtemperature for 2-3 h. Dicyclohexylurea (DCU) precipitate developed withtime.

Deprotection of Boc Group From BocNH-PEG-CO₂NHS

BocNH-PEG-CO₂NHS (3400 m.w., Shearwater Polymers, Huntsville, Al.) wasdissolved in 5 ml of CH₂Cl₂:TFA (4:1,v/v) solution. This mixture wasstirred at room temperature for 2 hours. TLC analysis revealed that thereaction had gone to completion. The product gave a positive ninhydrintest. The solvents were removed under reduced pressure and the sampledried under high vacuum.

Coupling FA to NH₂-PEG-CO₂,NHS

To the FAA reaction mixture, H₂N-PEG-CO₂NHS (186 mg, 0.057 mmole) wasadded in 2.5 ml of DMF:triethylamine (100:1 v/v) and the reactionmixture was stirred at room temperature overnight. TLC analysis showedthat the reaction had gone to completion. The product was UV andninhydrin positive. Solvents were removed from the reaction mixtureunder reduced pressure and the sample dried under high vacuum.

Coupling FA-NH-PEG-CO₂NHS to Polylysine

To a solution of FA-NH-PEG-CO₂NHS (100 mg, 0.027 mmole) in 5 ml ofDMF:Py (4:1 v/v), were added PLL (62.15 mg, 0.0224 mmole) and E_(t)3N(3.4 mg, 4.7 ul, 0.0336 mmole). The reaction mixture was stirred at roomtemperature overnight. At this time point TLC analysis revealed that anew polar spot was formed which was UV positive and gave a positiveninhydrin test. Solvents were removed from the reaction mixture underreduced pressure and the sample dried under high vacuum. The residualmaterial was suspended in CHCl₃ and spun at 4000 rpm for 30 min. Thesupernatant solid material was separated and washed with CHCl₃. PureSample weight 55.4 mg (37.5% Yield).

Conjugates of folate or PEG directly linked to polylysine or othercharged molecules may also be prepared by similar procedures.

b. Preparation of Folate-PEG-Polylysine (FA-PEG-GA-PL or FPGK) UsingGlutaric Acid

Preparation of Folic Acid Anhydride (FAA)

Folic acid (50 mg, 0.11 mmole) was dissolved in 6 mL ofN,N-dimethylformamide:pyridine (DMF:PY) (4:1 v/v). To this solution wasadded dicyclohexocarbodiimide (DCC) (140 mg, 0.68 mmole) and thereaction mixture was stirred at room temperature for ˜3 h. With timedicyclohexylurea (DCU) precipitate was observed in reaction mixture.

Coupling FA to PEG (3400)

To the FAA reaction mixture prepared above, H₂N-PEG-NHBoc (3400 m.w.Shearwater Polymers, Huntsville, Al.) (192 mg, 0.057 mmole) was added in1 ml of NN-dimethylformamide. This reaction mixture was stirred at roomtemperature overnight. At this point TLC analysis revealed that thereaction had gone to completion. The product formed was UV positive andgave a negative ninhydrin test. Solvents were removed under reducedpressure.

Deprotection of Boc Group From FA-NH-PEG-NHBoc

The residual material was dissolved in 5 ml of CH₂Cl₂:TFA (4:1 v/v) andthe reaction mixture was stirred at room temperature for 2-3 hr. At thistime point, TLC analysis revealed that the reaction had gone tocompletion. The product was UV positive and gave a positive ninhydrintest. Solvents were removed at reduced pressure and the residualmaterial dried under high vacuum.

Introduction of GA Molecule (FA-NH-PEG-NH-CO(CH₂);3000 H)

The residual material was dissolved in 5 ml of DMF:Py (4:1 v/v). To thissolution Et₃N (15.7 μl, 11.41 mg, 0.113 mmole) and Glutaric anhydride(9.7 mg; 0.085 mmole) were added and the reaction mixture was stirred atroom temperature overnight. TLC analysis showed that the reaction hadgone to completion. TLC analysis was performed in CHCl₃:CH₃OH:H₂O(65:30:5; v/v/v). The product gave a negative ninhydrin test. Theresidual weight of the sample was 649 mg.

Coupling Polylysine (PL) (FA-NH-PEG-NH-CO(CH₂)₃ CO-PL):

DCC (44 mg, 0.21 mmole) was added to a solution-ofFA-NH-PEG-NHCO(CH₂)3000 H (residual material) (162 mg, 0.043 mmole) in 4ml of DMSO:Py (4:1 v/v). This mixture was stirred at room temperaturefor 2-3 hrs. With time DCU precipitate developed. At this pointpolylysine HBr (32 mg, 0.011 mmole) was added to the reaction mixture in1.4 ml of DMSO:Py:Et₃N (1000:200:200 μl) solution. This reaction mixturewas stirred at room temperature overnight. At this time point TLCanalysis in CHCl₃:CH₃OH:H₂O (60:35:5 v/v/v) revealed that the reactionhad gone to completion. TLC analysis gave positive UV and ninhydrintests. Solvents were removed under reduced pressure. The residualmaterial was dissolved in CHCl₃ and the precipitate separated byspinning at 3000-4000 rpm for 30 minutes. The solid mixture wasseparated and washed with CHCl₃. The pure sample weighed 19.1 mg (27%Yield).

c. Preparation of Folate-PEG-Protamine (FA-PEG-GA-Pro or FPGPr) UsingGlutaric Acid

FA-PEG-GA was prepared as described in part b in theFolate-PEGPolylysine synthesis scheme.

Cougling Protamine to (FA-NH-PEG-NH-CO(CH₂)₃CO-Protamine):

DCC (8.03 mg, 0.039 mmole) was added to a solution ofFA-NH-PEG-NH-CO(CH₂)₃OOOH (residual material) (50 mg, 0.013 mmole) in3.0 ml of CHCl₃. This mixture was stirred at room temperature for 2-3hrs. At this point protamine free base (13.3 mg , 0.0033 mmole) wasadded to the reaction mixture in 1.5 ml of1,1,1,3,3,3-hexafluoroisopropanol. This reaction mixture was stirred atroom temperature overnight. At this time point TLC analysis inCHCl₃:CH₃OH:H₂O (60:35:5 v/v/v) revealed that the reaction had gone tocompletion. TLC analysis gave positive UV and ninhydrin tests. Solventswere removed under reduced pressure. The residual material was dissolvedin CHCl₃ and the precipitate separated by spinning at 3000-4000 rpm for30 minutes. The solid mixture was separated and washed with CHCl₃. Thepure sample weighed 5.5 mg (22% Yield).

Conjugates of folate alone or PEG alone directly linked to polylysine orother charged molecules may also be prepared by minor modifications ofthis method.

d. Preparation of Acetyl-PEG-Protamine (Ac-PEG-GA-Pro or AcPGPr) UsingGlutaric Acid

Several variations of the PEG conjugates were prepared as controls tocompare with the above conjugates. These were missing one of morecomponent of the active conjugate. The Ac-PEG-GA-Pro conjugate, orAcPGPr, was prepared by substituting an acetyl group for folate on theconjugate. It was, therefore, not targeted to a cell receptor.

In addition to control experiments, it may be desirable to use such anontargeted protecting moiety to modulate the liposomal surface foroptimal targeting in vivo. For instance, a non-targeted conjugate may beused in addition to a targeting conjugate on the liposomal surface.

Acetylation of NH2-PEG-NHBOC:

To a solution of H₂N-PEG-NHBOC (100 mg, 0.0294 mmole) in 3.0 mL of CHCl₃were added acetic anhydride (9.0 mg, 7.85 μL, 0.088 mmole) and excessamount of TEA. This reaction mixture was sealed under nitrogen andstirred at room temperature for 34 h. At this time point TLC analysis inCHCl₃:CH₃OH (9:1 v/v) revealed that the reaction had gone to thecompletion. TLC analysis gave negative Ninhydrin test. Solvents wereremoved under reduced pressure and residual material was used in nextstep without purification.

Degrotection of BOC gp from Ac-NH-PEG-NHBOC:

To a solution of Ac-NH-PEG-NHBOC in 2.2 mL of CH₂Cl₂ was added 0.25 mLof TFA. This reaction mixture was stirred at room temperature for 2 h.At this time point TLC analysis in CHCl₃:CH₃OH (9:1 v/v) revealed thatthe reaction had gone to the completion. TLC analysis gave positiveninhydrin test. Solvents were removed under reduced pressure andresidual material used in the next step without purification.

Introduction of GA Molecule on Ac-NH-PEG-NH,:

The residual material was dissolved in 3.0 mL of CHCl₃. To this solutionexcess amount of Et₃N and Glutaric anhydride (5 mg; 0.044 mmole) wereadded and the reaction mixture was stirred at room temperatureovernight. TLC analysis showed that the reaction had gone to completion.TLC analysis was performed in CHCl₃:CH₃OH (9:1 v/v). The product gave anegative ninhydrin test. The residual material was used in the next stepwithout further purification.

Coupling Ac-NH-PEG-NH-CO-(CH₂)₃-OOOH to Protamine (Pr):

To a solution of Ac-NH-PEG-NH-CO-(CH₂)₃OOOH in 2.0 mL of CHCl₃ was addedDCC (18.2 mg, 0.088 mmole) and the reaction mixture was stirred at roomtemperature for 2-3 h. At this time point white ppte was developed.Protamine (30.0 mg, 0.0074 mmol) in 1.6 mL of 1,1,1,3,3,3 Hexafluoroisopropyl alcohol was added to the reaction mixture and the reactionmixture was stirred at room temperature over night. At this time point,TLC analysis gave a polar positive spot. Solvents were removed underreduced pressure and residual material was suspended in CHCl₃. Thesuspension was centrifuged at 3500 rpm for 15 min. The solid materialwas separated from clear solution to give 23 mg, yielding 35%.

e. Characterization of the Amine Content of Protamine and PolylysineConjugates With PEG and Folate or an Acetyl Group

The ratio of protamine or polylysine to the remaining molecular mass inthe final products was characterized by fluorescent derivatization offree amino groups with fluorescamine, a reagent that becomes fluorescentupon reaction with amines. As a calibration of fluorescence response,weighed amounts of free polylysine or protamine were reacted withfluorescamine to yield a fluorescent product. Standard curves wereprepared for either protamine or the 3,000 molecular weight polylysinemolecule used for conjugate formation. The concentration of amines inthe conjugates was then determined by reaction with fluorescamine. Theassay was done in a 96 well Cytofluor 4000 fluorescence plate reader(PerSeptive Biosystems, Cambridge, Mass.). After dilution of the sampleinto 50 μL total volume of water, 200 μl of 130 mM NaBH₂O₃ buffer at pH9.5 and 50 pl 0.2 mg/ml fluorescamine which was dissolved in acetonewere added. The samples were incubated at room temperature for 20minutes after which the fluorescence was measured at 490±20 nm with395±12 nm excitation. The fluorescence readings were compared to thestandard curves to determine the amount of protamine or polylysine in agiven weight of conjugate and the ratio of this amount to the total masswas calculated. For the protamine conjugates with PEG and either folateor an acetyl group the expected ratio of a 1:1 conjugate is 0.52 and0.54, respectively, while determined ratios were 0.50±0.05 and0.56±0.05, respectively. For the polylysine conjugates FPK and FPGK, thecalculated ratios are 0.45 and 0.44, respectively, while the determinedratios were 0.64±0.17 and 0.35 t 0.05, respectively. Because of thepolydispersity of the polylysine used for this purpose, these ratios areonly approximate guidelines for the 1:1 complexes.

The protamine conjugates clearly contained a single protamine per PEGgroup, while the polylysine conjugates were close to the desired ratios.

f. Preparation of a Phospholipid Derivative of Folate and PEG,Folate-PEGDOPE (FA-PEG-DOPE or FPPE)

A phospholipid conjugate of folate and PEG was prepared to compare tothe conjugates of folate prepared above. The phospholipid conjugatewould insert hydrophobically into the liposomal membrane, while theother conjugates would interact with the liposomal membrane via anelectrostatic attraction. The results are further discusses in Example8.

Coupling DOPE to t-BOCNH-PEG-CO₂NHS:

To a solution of DOPE (20 mg, 0.026 mmole) in 3.0 mL of CHCl₃ were addedt-BocNH-PEG-CO₂NHS (91.5 mg, 0.026 mmole) and TEA (2.7 mg, 0.026 mmole).This reaction mixture was sealed under nitrogen and stirred at roomtemperature overnight. At this time point TLC analysis inCHCl₃:CH₃OH:H₂O (65:25:4 v./v) revealed that the reaction had gone tothe completion. Solvents were removed under reduced pressure andresidual material was purified on column chromatography to give 168 mg(53%). Some of characteristic ′H NMR (CDCl₃) signals are: 5 0.86 (t, 6H,J=6.35 Hz, Cl1₃), 1.25-1.27 (br, (CH₂),, for DOPE), 1.43 (s, t-BOC),3.63 (br, CH₂'s for PEG) and 5.32 (br signal for olefinic protons).

Deprotection of t-BOC from t-BOCNH-PEG-DOPE Conjugate:

To a solution of t-BOCNH-PEG-NH-DOPE conjugate in 4.0 mL of CH₂Cl₂ wasadded 1.0 mL of TFA. This reaction mixture was stirred at roomtemperature for 2 h. At this time point TLC analysis in CHCl₃:CH₃OH:H₂O(65:25:4 v/v) revealed that the reaction had gone to the completion.Solvents were removed under reduced pressure and residual material waspurified on column chromatography to give 44 mg. Some of characteristic′H NMR (CDCl₃) signals are: 8 0.86 (t, 6H, J=6.11 Hz, CH₃), 1.25-1.27(br, (CH₂)r, for DOPE), 3.63 (br, CH₂'s for PEG) and 5.32 (br signal forolefinic protons).

Coupling FA to NH₂-PEG-DOPE:

To a solution of FA (10.58, 0.026 mmole) in 3.0 mL of DMSO:Py (2:1 v/v)were added DCC (14.8 mg, 0.072 mmole) and NH₂-PEG-DOPE (45 mg, 0.012mmole). This reaction mixture was sealed under nitrogen and stirred atroom temperature for overnight. At this time point TLC analysis inCHCl₃CH₃OH:H₂O (75:35:6 v/v) revealed that the reaction had gone to thecompletion. TLC analysis gave positive UV, ninhydrin and molybdatetests. Solvents were removed under reduced pressure and residualmaterial was purified on column chromatography to give 21.5 mg (43%).Some of characteristic ′H NMR (CDCl₃:CD₃OD 8:1 v/v) signals are: 8 0.79(t, 6H, J=6.62 Hz, CH₃), 1.18-1.21 (br, (CH₂)_(n) for DOPE), 3.56 (br,CH₂'s for PEG), 5.25 (br signal for olefinic protons) and 6.60-8.6 (3signals for FA).

g. Coupling Antibody to Polylysine: Carbohydrate-Specific PolylysineCoupling to the Monoclonal IgG OVB-3

An antibody conjugate was also prepared for electrostatic linkage toliposomes to target such liposomes to the appropriate cells.

OVB-3 Isolation:

OVB-3 is a monoclonal (mab) IgG that binds specifically to human ovariancarcinoma cells. The hybridoma cell line was obtained from ATCC(HB-9147). Hybridoma cells were injected into Balb/c mice and asciteswas collected a couple weeks later. The mab was isolated from theascites using a Protein A column and stored in a phosphate buffer (150mM NaCl, 20 mM NaP, , pH 7.5)

Preparation of Thiolated Aolylysine:

Free sulfhydryl groups were introduced on to polylysine polymers bymodifying the amines with 2-iminothiolane (Traut's reagent).Hydrobromide polylysine in the 1,000 to 4,000 molecular weight range wasobtained from SIGMA. The polylysine (45 mg) was dissolved in 1.0 ml ofborate buffer (100 mM NaBH₂O₃ pH 8.0). To this solution, 1 ml of 95.9mg/ml 2-iminothiolane dissolved in water was added. The mixture wasincubated for 1 hour in the dark with gentle shaking. After incubation,the sample was transferred to a 30 ml glass centrifuge tube and 18 ml ofisopropanol was added. The sample was centrifuged for 20 minutes at 9KRPM. The supemate was removed and the pellet was dried with a gentlestream of N₂. The pellet was redissolved in 2 ml of the pH 8.0 boratebuffer.

Labeling the Polylysine with Alexa 350:

In order to provide a quantitative measure of the extent of mab topolylysine coupling, the thiolated polylysine was labeled with thefluorescent probe Alexa 350 carboxylic acid succinimidyl ester which wasobtained from Molecular Probes. A 10 mg/ml Alexa 350 DMSO stock solutionwas prepared. Seventy-five 0 of Alexa 350 stock solution was added to 2ml of previously prepared thiolated polylysine sample. The sample wasincubated in the dark at room temperature for 1 hour with gentleshaking. After incubation, the sample was transferred to a 30 ml glasscentrifuge tube and 18 ml of isopropanol was added. The sample wascentrifuged for 20 minutes at 9K RPM. The supemate was removed and thepellet was dried with a gentle stream of N₂. The sample was redissolvedin 100 to 200 gl of water. The sample was freeze-dried in order toobtain the sample weight. Following the determination of the sampleweight, the sample was dissolved in NaP, buffer (100 mM NaP, , pH 7.0)at a concentration of 20 mg/ml.

Oxidation of the Mab by Periodate:

The polylysine coupling to OVB-3 was done at the carbohydrate region ofthe mab. Two hundred p,l of 300 mM Na104 in water was added to 2 ml of 5mg/ml OVB-3 in 150 mM NaCl, 20 mM NaP, pH 7.5. The sample was incubatedfor 1 hour at room temperature in the dark. Glycerol ( 200 g,I ) wasadded to stop the oxidation reaction. The sample was then subjected to aPD-10 Sephadex G25 desalting column which was equilibrated with a pH 5.0acetate buffer (100 mM Na Acetate, pH 5.0). The sample wasreconcentrated back to 2 ml using an Amicon stirred ultrafiltration cellwith 30K MWCO filter.

Coupling the Thiolated Polylysine to the Oxidized OVB-3:

The thiolated polylysine was linked to the carbohydrate oxidized OVB-3using the heterobifunctional cross-linker MPBH(4-(4-N-maleimidophenyl)butyric acid hydrazide) which was obtained fromPierce. One hundred p,l of an 80 mM DMSO MPBH stock solution was addedto the previously prepared 2 ml oxidized OVB-3 solution (100 mM NaAcetate, pH 5.0). The final MPBH concentration was 4 mM. The sample wasincubated for 2 hours at room temperature in the dark with gentleshaking. The sample was then run down a PD-10 Sephadex G25 desaltingcolumn which was equilibrated with a pH 7.0 P; buffer ( 100 mM NaP1, pH7.0). The OVB-3 column fractions were pooled and 15 mg of previouslyprepared thiolated polylysine (100 mM NaP,, pH 7.0) was added. Thesample was incubated at room temperature for at least 3 hours. Theunreacted thiolated polylysine was separated from the OVB-3 polylysineconjugate by gel filtration on (Sephacryl-200 HR) or a Protein A column( Pharmacia Biotech HiTrap Protein A column) using a pH 7.4 TES buffer(150 mM NaCl, 10 mM TES, 0.1 mM EDTA, pH 7.4). The OBV-3 polylysineconjugate was pooled, reconcentrated by stirred ultrafiltration to astock solution concentration of about 1 mg/ml, and stored in therefrigerator.

EXAMPLE 3

Assay Methods and Materials:

a. Cell Cultures for Measurements of Liposome Binding and Transfection

OVCAR-3 cells were plated at 2×105 cells per ml in 96-well plates in 0.1ml per well of RPMI 1640 with 10% heat inactivated fetal bovine serum.Cells were allowed to grow for two days (approximately 4048 hours)before transfections were performed; at this point the cells were atconfluency. In the case of pHdependent liposomes described below, thecells were allowed to grow for only one day before transfection.

b. Measure of Cell Number (CBAM, Calcein Blue Acetoxy Methyl Ester)

Cell number in terms of total intracellular esterase activity wasdetermined by washing the cells with phosphate buffered saline (PBS),staining with calcein blue acetoxy methyl ester (CBAM-5 μM in PBS forapproximately 3045 minutes at room temperature), and then rinsing theplates with PBS). 100 μ/well of detergent solution (1% Cl₂E8, TE, pH 8)was added to each well. Cell number was determined in a Cytofluor 2instrument by determining the calcein blue fluorescence (excitation at360 nm, emission at 460 nm, gain usually at 80). Plates were againwashed 2 times with detergent solution and read again in the samedetergent solution to correct for background. A series of unlabeledcontrol wells were also read as an internal blank. All tansfection(EGFP) and liposome binding (rhodamine) fluorescent readings werecorrected by dividing by the CBAM readings.

c. Measurement of Binding of Liposomes to Cells

Liposomes comprising about 0.1 to about 1 mole % N-lissamine rhodamine Bsulfonyl) phosphatidylethanolamine were incubated with the cells and therhodamine fluorescence was observed visually and by measurement on aCytofluor II plate reader using 560 nm excitation and 620 nm emission.

d. Transfection Efficiency Assay

Transfection success, and expression of the transfected nucleic acid ina cell, can be detected in a number of ways, these generally dependingupon either detection of the physical presence of the nucleic acid inthe cell, e.g., by incorporation of radionucleotides in the nucleicacid, or by detecting expression of the protein encoded by the nucleicacid. This can be accomplished in a number of ways, including, withoutlimitation, where the protein is a detectable, e.g., fluorescent,marker, or where the protein is a selectable, e.g., cytotoxicagent-resistance, marker.

For example, the plasmid pEGFP-1 contains a DNA sequence encoding theenhanced green fluorescence protein, whose presence is detected byfluorescence microscopy or fluorescence plate reader. Accordingly,successful transfection of cells with this plasmid is readily determinedby assessing the quantity of fluorescence exhibited by the cells. Thetransfection activity of the liposomal preparations encapsulatingpEGFP-Cl plasmid DNA was tested as follows. Transfection solutions wereprepared by dilution of appropriate liposome or DNA samples into thedesired buffer or medium. The plates were aspirated to remove medium.

Transfection solutions (0.1 ml per well for 96-well plates) wereprepared by dilution of dialyzed samples containing the pEGFP-Cl plasmidinto medium or buffer (approximately 2 mM total lipid unless indicatedotherwise) containing 10% heat inactivated fetal bovine serum (unlessotherwise designated), and were then added to the wells and incubated at37 degrees C. for 3 hours. The wells were aspirated, and mediumcontaining 10% heat inactivated fetal bovine serum was added to eachwell. Because of the previously demonstrated silencing of transgenesunder the CMV promoter (Tang et al., 1997; Dion et al., 1997) a histonedeacetylase inhibitor, 5 mM sodium butyrate, included in the medium toenhance expression.

After incubation at 37 degrees C. in a cell culture incubator for 18-22hours, the medium was aspirated and a 0.5 ml wash of Dulbecco's PBS wasadded. At this point or just before the wash, photomicrographs weretaken of the samples still on tissue culture plates with an OlympusIMT-2 inverted microscope using the 10× objective. The samples were thendissolved in detergent and readings were taken for corrected total EGFPfluorescence, in terms of the total number of live cells, using thecalcein blue AM assay described above.

e. Beta Galactosidase Assay

Cells are assayed for β-galactosidase activity using thechemiluminescent β-galactosidase detection kit from Clontech accordingto the manufacturer's directions. Briefly, 200 NI washed IP cells arespun at 1200 rpm for two minutes. The supernatant is removed and thecell pellet lysed in 300 μ lysis buffer (100 mM potassium phosphate, pH7.8 with 0.2% Trition X-100, prepared as described by Clontech) byvortexing for -30 seconds and shaking gently for 15 min. Cell debris isthen removed by centrifugation at 14000 rpm for 2 min. 200 pl reactionbuffer is added to 45 pl cell lysate in 96 well plates, mixed andincubated for 60 min at room temperature. Luminescence is read on amicroplate luminometer (EG&G Berthold), recording light signals at 5second intregrals.

f. Sources of Materials

N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine(transesterified from egg PC), DOPC, EPC and N-Cl₂-DOPE were purchasedfrom Avanti Polar Lipids (Alabaster, AL). OVCAR3 ovarian carcinoma cellswere purchased from NCI-Frederick Cancer Research Laboratory (Frederick,Md.). The pEGFP-Cl plasmid, and E. coli DH5α competent cells werepurchased from Clontech Laboratories (Palo Alto, Calif.). pZeoSVLacZplasmid, competent cells and Hanahan's S.O.C. were purchased fromInvitrogen (San Diego, Calif.). Hanks Balanced Salt Solution (HESS),RPMI 1640 and heat inactivated fetal bovine serum were purchased fromGibco/BRL (Grand Island, N.Y.). DNase-free RNase and RNase-free DNase Iwere purchased from Boehringer Mannheim (GmbH, Germany). Agarose waspurchased from FMC Bioproducts (Rockland, Md.). Bacto agar, Bactotryptone and yeast extract were purchased from DIFCO Laboratories(Detroit, Mich.). Calcein blue acetoxy methyl ester (CBAM), PicoGreenand SybrGreen I dyes were from Molecular Probes (Eugene, Org.). Apolylysine-transferrin (pKT) and free polylysines were obtained fromSigma (St. Louis, Mo.). pKT consists of a 30-70 kDa poly-L-lysinecoupled to human holotransferrin such that there are approximately 3transferrin molecules per polylysine. There is also an fluoresceinisothiocyanate (FITC) group on each conjugate.

EXAMPLE 4

Charge Reversal Liposomes: Electrostatic Linking of Target!ng/Protecting Modules to Charge Reversal Liposomes is pH-Dependent.

In one embodiment of the modular delivery invention, liposomes weredesigned to reverse charge on a change from pH 7 to a lower pH found incell organelles such as endosomes. To determine whether thetargeting/protecting conjugates would stably bind to liposomes underconditions of physiological pH and be released from such liposomes underconditions such as lower pH, the following experiment was carried out.Liposome were made by first mixing POPE:DOMDODAP:Cholesterol:Cholesteryl hemisuccinate:Oleoyl acteate(51:12.2:10.7:10.7:2.6:12.8:10.7) in chloroform. EGFP plasmid, condensedwith spermine, was encapsulated during liposome preparation according tothe twostep emulsion method (example 1). 300 mM sucrose was used asplasmid and spermine dilution buffer. 300 mM sucrose was used in theinitial hydration buffer to facilitate separation of the liposomes frombulk buffer by centrifugation. The final liposome preparation wasdialyzed against PBS before use. The final lipid concentration was ˜20mM.

Cell-targeting protein complexes were bound to the external surface ofthe plasmid-containing liposomes through non-covalent interactions. AtpH 7.4, the liposomes have a net negative charge that provides a bindingsurface for positively charged proteins or moieties linked to positivelycharged polypeptides (pKT, pK-OVB3-Ab). The number of liposomes in a 200NJ aliquot was estimated assuming a 20 mM total lipid concentration anda liposome diameter of 150 nm. Ratios of 1-5 protein complexes perliposome were tested. Protein stock solutions (FITC-labeled pKT,pK-OVB3) were made at a concentration of 0.5 mg/ml in PBS, pH 7.4. Analiquot containing the desired amount of protein was then diluted in 200μPBS. For a standard protein binding assay, the protein solution wasadded dropwise to the liposome solution with continual mixing. Equalvolumes of protein solution were mixed with the liposomes at mole ratiossuch that the overall charge of the liposome-protein assembly remainednegative. The protein/liposome mixture was incubated at room temp for15-30 min. and then separated into two equal volume aliquots. The pH ofone aliquot s was lowered to pH 5 by addition of HCl, followed byincubation at room temp for an additional 15 min. Liposomes and boundprotein were separated from free protein by centrifugation at 14,000 gfor 30 min. The liposome pellets were resuspended in PBS and the pH ofall supernatants returned to pH 7.4 with addition of NaOH beforeanalysis. Protein levels in the supernatant were measured using theBiorad Coomassie protein assay for pK-OBV3-Ab. For the FITC-labeled pKT,fluorescence was measure for each fraction (Ex:485 Em:530). Liposomeswithout added ligand and protein solutions without added liposomes wereused as controls.

Table 1 shows the results of the pH-dependent binding of pKT at twoconcentrations, 2 or 5 pKT complexes per liposome. (Note: halfneutralization of the liposomal negative charge would occur with −3 pKTbound.) At pH 7.4 virtually all of the protein pelleted with theliposome fraction during centrifugation at both pKT concentrations. Whenthe pH was lowered to 5, a difference was found between the samples. Ata pKT/liposome mole ratio of 2, all of the pKT remained associated withthe liposome even at low pH. At the higher pKT/liposome ratio, afraction of the pKT completely dissociated from the liposome at pH 5 andremained in the supernatant after centrifugation. These results suggestthat there may be two types of binding sites for pKT on the liposome: Ahigher affinity site that is not pH-dependent and binds up to two ligandcomplexes per liposome and a lower affinity site where the proteincomplex can be toggled on or off the liposome surface by a change in pH.

Most of the pKT (-80%) remains bound to these liposomes in 20% serum,and a significant amount remains bound up to 40% serum. This amount oftargeting ligand is sufficient to provide specific cell binding througha transferrin receptor.

Table 1 also shows the pH-Dependent binding of 3K-polylysine conjugatedto an antibody to the OVB3 antigen, which is found on ovarian cancercells (pK-OVB3). Analogous to the pKT study, 2 or 5 pK-OVB3-Ab complexeswere bound through electrostatic interactions per negatively chargedliposome. The liposomes and bound targeting ligand were separated fromunbound soluble protein by centrifugation. The amount of unboundpK-OVB3-Ab in the supernatants increased significantly when the pH waslowered from 7.4 to 5 (˜30% increase at a pK-OVB3-Ab/liposome mol ratioof 2, and ˜50% increase at a mol ratio of 5).

Taken together, these results demonstrate that these liposomes bind avariety of targeting ligands via a cationic linker at neutral pH. At lowpH, the affinity of the ligand complex may vary with the polyvalency ofthe cation, and at least some of the targeting complex should dissociatefrom the liposome at low pH. At pH 5, as in the endosomal or lysosomalcompartment, the charge on cholesterol hemisuccinate should change fromnegative to neutral, and DODAP would become fully positively charged,resulting in a net positively charged membrane surface. This surfacewould have less affinity for the positively charged polylysine. TABLE 1pH-Dependent binding of targeting modules to charge-reversal liposomesof total protein added found free in bulk buffer' Ligandpolylysine-transferrin^(b) polylysine-OVB3-IgG{grave over ( )} Ligandpolylysine- polylysine- mol ligand per transferrin^(b) OVB3-IgG^(c) molliposomes pH 7.4 pH 5 pH 7.4 pH 5 2 1.5 0 0 30 5 1 18 7 33^(a)Free and liposome-associated ligand complexes separated bycentrifugation.^(b)Fluorescence of FITC-labeled ligand measured for isolated liposomepellets and supernatants.^(c)Protein level measured in isolated supernatants.^(d)HCI added to sample at 7.4 to reduce the pH

EXAMPLE 5

Charge Reversal Liposomes: Binding of Serum Proteins is Minimal

Effect of Ligand Complexes on C3 Acitvation by Liposomes:

There are many factors that influence the circulation lifetime ofliposomes in vivo. One critical aspect is the binding/activation ofcomplement factor C3 by the liposomes (see Semple, et al. (1998)Advanced Drug Delivery Reviews 32:3 17 and Devine and Bradley (1998)Advanced Drug Delivery Reviews 32:19-29 for reviews); activation of C3increased greatly liposome clearance from blood. The effect of modulartargeting complexes on the activation of C3 by liposomes was testedusing the pH-sensitive formulation.

Binding of C3Complement Component to Liposomes:

The ability of the liposomes to bind the C3 component of complement wastested by a modification of the in vitro hemolysis assay of Devine etal. (1994) Biochim. Biophys. Acta 1191:43-51 and AN et al. (1997)Biochim. Biophys. Acta 1329:370-382. Briefly, antibody-sensitized sheeperythrocytes (all reagents were from Sigma Chemical Co., St. Louis, Mo.,unless noted) were washed and resuspended as directed by themanufacturer. Rat sera complement was hydrated in water then diluted2-fold with GVB²⁺buffer. 200 μ liposomes, with or without boundtargeting ligand (polylysine-transferrin, polylysine-anti-OVB3-IgG orpolylysine-PEG-folate) were mixed with 100 ml diluted rat sera andincubated at 37° C. for 30 min with continuous shaking. 300 μGVB²⁺buffer was added and any liposome aggregates pelleted by centrifugation.Eight successive 2-fold dilutions, into GVB 2+ buffer, were then donefor each sample. 100 μ of the sheep erythrocytes were added to an equalvolume of each liposome/sera dilution and incubated for 30 min at 37° C.with shaking. Further hemolysis was stopped by adding GVB-EDTA buffer.Intact red cells and membrane fragments were then pelleted, and theabsorbance of the supernatant was measured at 415 nm. GVB²⁺ bufferserved as a negative control for C3 binding, and the absorbance of anequal volume of osmotically lysed red cells taken to be 100% hemolysis.

C3-mediated hemolysis curves were generated for the charge reversalliposomes with and without bound targeting ligand. The charge reversalliposomes alone gave a hemolysis profile very similar to the buffercontrol, suggesting little or no activation of C3 by these liposomes.When targeting complex was added, the hemolysis profile was similar tothe liposome-only curve for all three targeting modules tested. Thedecrease in CH50 relative to buffer was determined from linear fits ofthe middle portion of the hemolysis curves by the method described by AMet al. These results are shown in Table 2. Very minor decreases (3.3-7%)in CH50 values were found for the pH-sensitive liposomes with andwithout bound targeting ligand. These values are significantly less thanthose observed by Ahl, et al. (1997) for liposomes that were rapidlycleared from the circulation of rats. They are also less than valuesfound by those investigators for liposomes not cleared completely fromthe circulation in the first hour.

These results suggest that the presence of bound targeting moleculesdoes not significantly increase the amount of liposome-mediated C3activation. This lack of C3 activation may result in increasedcirculation time for these liposome vectors. TABLE 2 Effect of targetingligand on complement fixation by pH-sensitive liposomes Targeting ligand% decrease in CH50^(a) no ligand 3.7 ± 1.2 (3)polylysine-transferrin^(b) 3.3 ± 1.7 (4) polylysine-antiOVB3^(c) 4.5 ±1.3 (4) polylysine-PEG-folate^(d) 7 (1)^(a)% decrease relative to buffer (100% CH50, 0% decrease) calculated asdescribed by Ahl et al. The number of experiments is listed inparentheses.^(b)combined data for samples with 2-5 mol pK-transferrin per molliposomes^(c)combined data for samples with 2-5 mol pK-anti-OBV3 per molliposomes^(d)single sample with 5 mol pK-PEG-folate per mol liposome

EXAMPLE 6

Charge Reversal Liposomes: Enhancement of Transfection Efficiency byTargeting

NIH Ovcar-3 cells were grown as in Example 3 for 24h before transfectionassays were performed. Aliquots of pH-sensitive liposomes containingpEGFP-C1 plasmid were mixed with a desired amount of targeting ligand inPBS and incubated at room temperature for 15 min. The sample was diluted5-fold into RPMI with or without heat inactivated FBS and 50 of a 50 mMCaCl₂/20 mM MgCl₂ solution added per 0.5 ml liposomes and mixed. 90 μliposome solution was added to PBS-washed cells in each well andincubated for ˜5 h at 37° C. The transfection solution was removed, thecells washed with PBS, then allowed to incubate for 24-36 h in RPMIsupplemented with 10% heat inactivated FIBS and 5 μM sodium butyrate.

EGFP transgene expression was determined qualitatively by viewing thecells still in the culture plate by epi-fluorescence microscopy andquantitatively by reading the fluorescence intensity of each well afterlysing the cells in 1% Cl₂E8 detergent using a PE Biosystemscytofluor4000 platereader (Ex:485 Em:530) as in Example 3. Cellviability was measured by an enzymatic fluorescent assay. Prior tolysis, the cells were incubated for 40 min in 25 p,M calcein blue AM(Molecular Probes). Fluorescence intensities of the calcein bluehydrolysis product (Ex:360, Em:460) were measured during the same run asthe EGFP readings. For comparison between conditions, transgeneexpression was corrected for cell number by dividing the EGFP reading bythe calcein blue reading for each well.

The ability of the pH-triggerable liposomes to transfect cells wastested in Ovcar3 cells in vitro. Three targeting ligands were tested(pKT, pK-OVB3 and FPK) at 1, 3 or 5 protein complexes per liposome. Theliposome formulation, alone or in combination with the targetingligands, showed a small amount of cell toxicity. Calcein blue levelswere approximately 20% lower for wells incubated with theplasmid-containing liposomes than for untreated cells or cells incubatedovernight in media with sodium butyrate but without liposomes. Theamount or type of targeting moiety did not significantly alter the levelof calcein blue measured.

Transgene expression was first assessed qualitatively by epifluorescencemicroscopy. FIG. 1 shows representative photomicrographs of Ovcar3 cellstransfected with the pH-sensitive charge reversal liposomes withpEGFP-Cl plasmid encapsulated. Without bound targeting ligand, low, butclearly visible cells filled with EGFP were observed (FIG. 1 a). Withthe addition of pKT, the number of cells showing green fluorescenceincreased (FIG. 1 b). The absolute number of cells transfected variedsomewhat from one transfection experiment to another. However, therelative amount of transfection, when comparing different targetingmoieties, was consistent between experiments. Similar results wereobtained with pK-OVB3 and FPK ligands.

The effects of increasing amounts of cell targeting moiety weredetermined quantitatively for pKTor FPK ligands. The results are shownin FIG. 2. The amount of transgene expression was corrected for cellnumber by dividing the EGFP fluorescence reading by the calcein bluereading for each well after background subtraction. This value was thenmultiplied by 1000 for plotting. An increase in transgene expression wasfound with increasing ligand concentration for all three bindingtargeting agents. The higher transgene expression for the pKT series,may simply reflect the increased number of targeting ligands per proteincomplex. pKT has 3 transferrins per polylysine chain wherepolylyssne-PEG-folate has 1 ligand per polylysine chain. The titrationof targeting complex was stopped at a 5:1 protein: liposome mol ratio tokeep the overall charge of the liposomes with pKT and pK-folate negativeat pH 7.4. As a control, pure 30K polylysine was used as targetingligand at a 3:1 protein: liposome ratio. The amount of transfection wassimilar to that for no ligand.

EXAMPLE 7

Fusogenic AfLacyl-PE Liposomes: Demonstration of Enhancement ofTransfection Efficiency Mediated by Folate Targeting.

Fusogenic N-acyl-PE liposomes are negatively charged at neutral pH asare the charge neutralization liposomes. The charge density of theN-acyl-PE liposomes decreases at lower pH, but does not completelyreverse. However, partial neutralization can also allow some looseningor exchange of electrostatically bound conjugates at low pH. Results andfurther discussion are provided in Example 8.

Liposomes composed of 70% NCl₂DOPE and 30% DOPC encapsulating spermineand the pEGFP-Cl plasmid were prepared as in example using the two stepemulsion method followed by pelleting and washing of the liposomes toremove external DNA. A stock of liposomes 20 mM in total lipidconcentration was mixed with the appropriate amount of a 340 p,M stockof folate-PEG-glutarylprotamine (FPGPr) conjugate or a controlderivative in which the folate group was replaced by an acetyl group(AcPGPr). The mixture was diluted with Hanks buffered salt solution(HBSS) without Ca²⁺, or Mg²⁺ to reach a total lipid concentration of 4mM and heat inactivated fetal bovine serum was added to a finalconcentration of 10% (v/v). To adjust the Ca^(t)+ and Mg²⁺concentrations to near the expected physiological levels a stock of 60mM CaCl₂ and 40 mM MgCl₂ was added at 20 μ per ml of liposome solutionjust before placing the liposomes into the empty wells of the 96-welltissue culture plates. Plates were prepared and transfection analysiscarried out as in Example 3.

The data are shown in FIG. 3. The folate conjugates greatly enhancedtransfection efficiency of the liposomes, while a control conjugatebearing only an acetyl group did not. These data demonstrate thattransfection by these liposomes is enhanced by electrostatically boundtargeting/stabilizing conjugates and that the effect is dependent on thepresence of folate in the conjugate. This suggests that a folatereceptor is utilized.

EXAMPLE 8

Fusogenic WLacyl-PE Liposomes with Folate Targeting: ElectrostaticallyBound Targeting/Stabilizing Modules Versus Covalent Coupling to Lipids.

Liposomes as described in Example 7 were prepared for association withseveral conjugates containing folate, PEG and a positively charged groupfor interaction with the membrane. These conjugates werefolate-PEG-protamine with a glutaryl linker (FPGPr),folate-PEG-polylysine with a glutaryl linker (FPGK) andfolate-PEG-polylysine (FPK) synthesized as described in the Assays andMethods section. A folate-PEG-phosphatidylethanolamine (FPPE) derivativewas also synthesized for comparison to the conjugates that interact byelectrostatic interaction. The FPPE derivative inserts into theliposomal membrane and is held there by strong hydrophobic interactions.The other derivatives bind to the liposomes via an electrostaticinteraction and can potentially dissociate in the endosome so that thefusogenic membrane can be exposed. The FPPE cannot dissociate as easily.The dissociation is important to remove the large PEG-containingtargeting moiety for membrane fusion to occur.

Liposomes were prepared as described in Example 3, containing 200 mMsucrose with spermine and DNA. After pelleting and washing to removeexternal DNA, the various conjugates were associated with the liposomes.For the FPPEcontaining liposomes, FPPE was incorporated into theliposomes at 2.5 mole % of the total phospholipid. The FPGK, FPK andFPGPr conjugates was added to the external leaflet of the liposomes at aconcentration that also was 2.5 mole % of the external phospholipid.Buffer, serum and divalent cations were added to the samples asdescribed in example 7 before addition to OVCAR-3 cells in 96-wellplates. The final total lipid concentration was 4 mM. After incubationsas described Example 7, the green fluorescence due to the expression ofEGFP was determined as described in Example 3.

FIG. 4 shows the comparison between the various conjugates in that theelectrostatically-linked conjugates were much more active than thecovalentlylinked FPPE conjugate. Similar results were obtained in whichthree separate preparations of the FPPE-containing liposomes werecompared to the FPGPr conjugate. The FPGPr conjugate was also comparedto the FPPE conjugate at a lower mole percentage in the membrane (0.4mole %). In this case a similar result was obtained, i.e. that the FPPEconjugate showed no transfection activity, while the electrostaticconjugate showed substantial activity. The FPPE conjugate was notentirely inactive as it did show some enhancement of transfectionactivity with the charge reversal liposomal system (examples 4-6) at avery low mole percentage (less than 0.1 mole %).

EXAMPLE 9 Fusogenic Af-acyl-PE Liposomes: Targeting Via the pK-OVB-3Antibody Conjugate

Another embodiment of the modular targeting/stabilizing concept involvesan antibody to ovarian cancer cells called OVB-3 coupled to linkers asin Example 2.

The pK-OVB-3 conjugate was found to bind to anionic liposomes, but notto zwitterionic liposomes, indicating the electrostatic nature ofbinding. This was demonstrated with more than one type of anionicliposome (data not shown).

Stability of Ligosomal Membrane

The effect of pK-OVB-3 targeting module binding on liposome integritywas examined by fluorescent NBD-phospholipids. NBD labeledphosphatidylethanolamine (headgroup label, transesterified from egg PC)was obtained from Avanti Polar Lipids (Alabaster, Al.) and incorporatedinto /V-acylPE liposomes (example 1) at 0.5 mole % of total lipid. Themembrane impermeant ion dithionite has been shown to rapidly reduce theNBDphospholipid in the outer lipid monolayer to a non-fluorescent form.The remaining NBD-phospholipid in a sealed liposome can only be reducedfollowing the addition of a detergent. The addition of 20 mM sodiumdithionite to NBDphospholipid labeled N-Cl₂-DOPE /DOPC (7:3) liposomescontaining encapsulated DNA (prepared as in example 1) rapidly decreasedthe level of NBD fluorescence from the outer monolayer as shown in FIG.5. An approximate reduction of 60% indicates that a single bilayermembrane is found in most of the liposomes. Addition of detergentreduced the remaining NBDphospholipid on the inner monolayer of theliposomes by dissolving the liposomes. pK-OVB3 conjugate binding tothese liposomes had no significant effect on the magnitude or timecourse of the NBD-phospholipid reduction by sodium dithionite. Thisindicates that conjugate binding to the liposome surface did notsignificantly destabilize or alter the basic structure of thesefusogenic liposomes.

Enhanced Uptake of Liposomes by gK-OVB-3:

Liposomes with pK-OVB-3 targeting modules were prepared by incubatingnegatively-charged liposomes with the pK-OVB-3 conjugate for 15 minutesat room temperature. During this incubation essentially all the pK-OVB-3targeting modules are electrostatically bound to the surface of theanionic liposomes. Liposomes with pK-OVB-3 targeting modules wereprepared at conjugate to lipid ratios ranging from 0 to 100 μ proteinper μmole lipid. These pK-OVB-3 coated liposomes were then immediatelyincubated with OVCAR-3 cells at a lipid concentration of 1 mM for 1½hours at 37° C. in the presence of 10% heat inactivated fetal calfserum. The OVCAR-3 cells were grown in Costar 96 well plates forapproximately 24 hours starting at a cell concentration of 2×10⁴ cellsper well. Following this incubation the OVCAR-3 cells were extensivelywashed by aspiration to remove unassociated liposomes. The liposomeswere labeled with 0.2 mole percent of the fluorescent lipidrhodamine-DOPE to follow the liposome uptake in a fluorescent platereader (560±10 nm excitation, 520 t 20 nm emission). FIG. 6 shows thatincreasing the amount of pK-OVB-3 targeting module bound to the surfaceof N-Cl₂-DOPE/DOPC (7:3 mole ratio) liposomes significantly increase thelevel of OVCAR-3 cell uptake of these liposomes in the presence of 10%serum.

Enhancement of Transfection by OVB-3 Targeting:

A series of transfection experiments were performed to test the effectof the antibody conjugates. The pK-OVB-3 conjugates were prepared asdescribed above. A stock of polylysine-OVB-3 antibody conjugate (conc.0.5 mg/ml), a conjugate of a 3K polylysine to the OVB-3 antibody, wasprepared as described above.

N-Cl₂-DOPE/DOPC ( 70/30) liposomes were prepared to encapsulate theplasmid pEGFP-Cl, spermine and sucrose as described in Example 1 andpelleted and washed as described in Example 1. OVCAR-3 cells were grownin 96 well plates as described in Example 3.

All transfection experiments were with OVCAR-3 cells in tissue culture,as described above. Varying amounts of the OVB-3 conjugate were addeddirectly to a 40 mM liposome stock and diluted into HBSS without calciumor magnesium. The final total lipid concentration was 4 mM for allsamples. 10% heat inactivated fetal bovine serum was added to eachsample. 20 p,l of a 60/40 mM Ca/Mg stock was added to every ml ofliposome solution just before addition to tissue culture plates.Experiments were performed in quadruplicate.

Liposomes were incubated for transfection of OVCAR-3 cells as describedabove. Total cell number and transfection activity were assessed byfluorescence measurements as described above.

The results are shown in FIG. 7. In FIG. 7, the fluorescence of EGFPcorrected for total cell number by the calcein blue reading was plottedagainst the concentration of the OVB-3-polylysine conjugate. Thecorresponding fluoresence photomicrographs are shown in FIG. 8. As canbe seen in the Figures, the antibody-polylysine conjugate greatlyenhances transfection efficiency in 10% serum.

Specificity of Enhancement of Transgene Expression for the OVB-3Antibody:

The monoclonal antibody OVB-3 had a relatively high level of binding toOVCAR-3 cells, while non-specific mouse serum IgG binding to OVCAR-3cells was relatively insignificant. Plasmid encapsulated N-Cl₂-DOPE/DOPC(7:3 mole ratio) liposomes were prepared with pK-OVB3 or mouse serumpK-IgG targeting modules as desceibed above. There was no significantdifference in the level of polylysine incorporation for the two antibodyconjugates. The conjugate to lipid ratio for both liposome preparationswas 12.5 μg protein per gmole lipid. The fusogenic liposomes were thenincubated with OVCAR-3 cells for 3 hours at 37° C. at 4 mM lipid in 10%heat inactivated serum. This medium was replaced by growth medium andthe cells were allowed to grow overnight. GFP transgene expression wasdetermined by GFP fluorescence (485±10 nm excitation, 530±12 nmemission) and was normalized to the total number of living cells using acalcein blue AM fluorescence assay. The targeting module made withOVCAR-3 specific monoclonal antibody OVB-3 significantly increased GFPtransgene expression of N-Cl₂-DOPE/DOPC (7:3 mole ratio) liposomesrelative to control non-targeted liposomes, while the non-specific mouseIgG-pK conjugate did not produce any significant increase in transgeneexpression. The s relative amounts of transgene expression (normalizedto 100 for the OVB-3 results) were 100±21 for the OVB-3 targeted systemversus 23 t 5 for no targeting and 20±15 for the non-specific IgGtargeted liposomes. This demonstrates the target specific characteristicof the OVB3-pK targeting module.

EXAMPLE 10

Fusogenic 1VLacyl-PE Liiposomes: Targeting via Polylysine-Transferrin(pKT)-Enhancement of Binding to OVCAR-3 cells

Liposomes were prepared with approximately 1 mole % Lissaminerhodamine-phosphatidylethanolamine as a fluorescent membrane probe.Liposomes prepared as described in Example 1 ( 70/30 NCl₂DOPE/DOPC) wereincubated with varying concentrations of polylysine transferrin (pKT).The pKT was made up as a 1 mg/ml solution in Hanks balanced Saltsolution (HBSS) without Ca or Mg. Since aggregation of the liposomesoccurred at a ratio of approximately 0.3 g conjugate (pKT)/mmole oftotal phospholipid (the expected range for charge neutralization at theouter liposome surface for liposomes of this composition), pKT/liposomeratios were kept below this range for the investigations.

In order to determine if the pKT conjugate affected the binding ofliposomes to OVCAR-3 ovarian cancer tumor cells, cells grown in 96 wellplates were incubated with liposomes with and without the pKT conjugateelectrostatically bound to the liposome. A ratio of approximately 0.2 gpKT/mmol lipid was used in all samples. Cells were incubated withliposomes at 0.1 mM, 1.0 mM, 5 mM and 15 mM total lipid in the presenceand absence of serum (heatinactivated FBS at 10%) to determine theeffect of serum on binding of the liposomes to the cells. The bindingsolutions were prepared by adding the materials in the following order:liposomes, pKT, buffer, serum where the buffer is HBSS without withoutCa²⁺, or Mg²⁺. Just before addition of the solutions to the cells, Ca²⁺and Mg²⁺ were added to adjust the overall concentrations to 1.2 and 0.8mM respectively. At the termination of the incubation period, the wellswere aspirated, rinsed to remove unbound liposomes and binding wasdetermined by the amount of rhodamine fluorescence bound to the cells.The results are shown in FIG. 9. Liposomes having no polylysinetransferrin bound to their bilayer showed limited binding to the cellsurface when incubated in the presence of serum. However, when theliposomes had polylysine transferrin bound to their surface, the bindingto the cells was significantly increased. In the absence of serum, thepolylysine transferrin conjugate had little effect on the liposomebinding to OVCAR 3 cells. The serum proteins significantly inhibit thebinding to liposomes to the cells. Transferrin pK conjugate attached tothe liposomes reduced or eliminated the serum induced inhibition ofliposome binding to the cells.

It is clear from the results that the addition of pKT greatly enhancesbinding of liposomes to these cells. Further, the pKT appears to reduceor eliminate the serum inhibition of liposome binding.

EXAMPLE 11

Fusogenic N-Acyl-PE Liposomes: Enhancement of Binding and Transfectionby Polylysine-Transferrin Targeting

N-Cl₂-DOPE/DOPC liposomes containing spermine-condensed pEGFP plasmidDNA-liposomes and Labeled by 0.5 mole % lissaminerhodaminephosphatidylethanolamine were prepared. In one experiment,sucrose-loaded liposomes were prepared by inclusion of 200 mM sucrose inthe buffer with DNA and the buffer with spermine. After removal oforganic solvent and formation of liposomes and dilution into 300 mMsucrose buffer, the liposomes were extruded and dialyzed into HanksBuffered Salt Solution without Ca²⁺ and Mg²⁺ (HBSS). The liposomes werethen pelleted, washed and resuspended at twice the normal concentrationin HBSS, i.e., to a final lipid concentration of approximately 40 mM.This stock was diluted to 20 mM and was used for experiments in 96 wellplates

As described in Example 3, 96 well plates were seeded with OVCAR-3 cellsat 2×10⁵ cells/ml of medium (RPMI 1640 with 10% fetal bovine serum) twodays prior to transfection experiments. Transfections were performed, asdescribed in Example 3, by evacuating the wells and adding 100 Al/wellof the desired premixed transfection solutions and incubating at 37° C.for 3 hours. At this point, the transfection solutions were removed and100 μ/well of RPMI 1640 medium with 10% FBS and 5 mM sodium butyrate wasadded. After overnight incubation at 37° C., the plates were treated forfluorescent assay of transfection as described in Example 1. Plates werewashed 2× with the same detergent solution and read again in the samedetergent solution. All EGFP readings were corrected by dividing by theCBAM readings for total cell esterase activity.

In parallel, a set of experiments was performed using empty liposomeslabeled with a lissamine rhodamine-PE derivative (ex. 560 nm, em. 620nm),. These were the same liposomes as described in Example 1. Theliposomes served as indicators of liposomal binding under variousconditions and as controls, especially in the cases where pKT was used.In the latter case, the fluorescein labeling of the pKT is a potentialinterference in the EGFP readings for transfection, and appropriatecorrections were made in some cases. In comparison to the EGFPplasmid-containing liposomes, the rhodamine labeling level was higher,and the diameter was slightly smaller for these liposomes.

All experiments, including binding experiments, were performed in themanner of a transfection experiment, i.e. a 3 hour incubation withliposomes, followed by overnight incubation with serum andbutyrate-containing medium.

Comparisons were made at a single liposome concentration and in 10% FBS,while other components varied. The order of addition of materials was aslisted below. The added “buffer” was HBSS without Ca/Mg in all cases.Ca²⁺and Mg²⁺were added at the end as described above. Liposome stockswere approximately 20 mM total lipid.

Results are shown in FIG. 10. FIG. 10 demonstrates that pKT enhances thebinding of liposomes to cells and transfection in a dose dependentmanner. In FIG. 11, photos of the same experiments also demonstrate thepKT effect, where red fluorescence indicates liposomal lipid and greenfluorescence the expression of the delivered gene.

EXAMPLE 12

Fusogenic IVLacyl-PE Liposomes: Inhibition of Polylysine TransferrinMediated Transfection by an Anti-Transferrin Antibody

The effect of an anti-transferrin antibody was further investigated inthis example. Liposomes and assays were prepared as described inExamples 1 and 3. Comparisons were made at 1 mM total lipid, 0.2 mg/mlpkT and in 10% FBS (always heat inactivated as in all previousexamples). The samples also contained either 1.4 mg/ml anti-transferrinantibody (Sigma, St. Louis) or 1.4 mg/ml nonspecific bovine IgG (Sigma,St. Louis). Materials were added in the order described in Example 7 toprepare stocks for addition to tissue culture wells. Ca², and Mg²⁺ wereadded at the end as in Example 7.

Photos were taken the day after the transfection incubation, and theresults are shown in FIG. 12.

FIG. 12 clearly shows that the anti-transferrin inhibited transfectionby the liposomes. It therefore appears that this antibody inhibitstransfection mediated by the polylysine-transferrin conjugate, althoughbinding of the liposome to cells is not strongly inhibited.

Notably, polylysine alone at a concentration approximately equal to thepKT concentration did not mediate either liposome binding ortransfection (see Example 13). Therefore, it appears that thetransferrin molecule plays some role in the binding of the liposomes tothe cells, and undoubtedly plays a role in enhancing transfection.

Example 13

Fusogenic AfLacyl-PE Liposomes: Polylysines Alone Do Not MediateLiposomal Delivery and Transfection.

The OVB-3-polylysine conjugates were prepared as described above. ThepK-OVB-3 was added as a 0.5 mg/ ml stock to a 40 mM lipid liposomesolution and then diluted with HBSS without Ca/Mg such that the finaltotal lipid concentration was 4 mM and the pK-OVB-3 concentration of 0.1mg/ml. The weight percent of polylysine (pK) in the conjugates wasestimated to be 3%. Based on this estimate, a stock of 3 kDa pK atapproximately 0.015 mg/ml was used at equal volume as the pK-OVB-3solutions to prepare liposome conjugates, i.e. the final pKconcentration was 3% of the pK-OVB-3 or 0.003 mg/ml.

The pKT sample was obtained from Sigma (St. Louis) and is describedabove. It contains 30-70 kDa polylysine group. For comparison to thisconjugate, a 30-70 kDa polylysine (not conjugated) was obtained fromSigma (St. Louis). Based on the manufacturer's determination ofapproximately 0.3 polylysine (pK) units per transferrin in theconjugates, the appropriate amount of free pK was added to the liposomesin the comparison experiments. In the pKT experiments, the liposomeconcentration was at 2 mM total lipid and the pKT concentration 0.1mg/ml. The liposome-pKT complexes were prepared by adding theappropriate amount of the 1 mg/ml pKT stock to a concentrated (20 mMtotal lipid) liposome solution and then diluting with the appropriateamount of HBSS without calcium or magnesium.

For the FPK conjugate experiments, 40 μl of a 340 μ/ml stock of FPK wasadded to 40 μ of 40 mM liposomes and then diluted to give a final lipidconcentration of 4 mM and a final FPK concentration of 34 μg/ml.

For FPGK conjugate experiments, 80 gl of a 340 μg/ml stock of FPGK wasadded to 40 p.1 of 40 mM liposomes and then diluted to give a finallipid concentration of 4 mM and a final FPK concentration of 68 μ/ml.

The FPK and FPGK conjugates also contain 3 kDa polylysine (pK) units.For comparison of pK to the FPK and FPGK conjugates, a stock of 3 kDa pKequivalent to the FPK and FPGK solutions was used. Since the pK isapproximately 40 weight % in these conjugates, a 136 μ/ml stock wasused. The final lipid concentration in the transfection experiments was4 mM and the final FPK and FPGK concentrations were 34 and 68 mg/mlrespectively, while the final free pK concentration for comparison was27 p.g/ml.

N-Cl₂-DOPE/DOPC ( 70/30) liposomes were prepared to encapsulate theplasmid pEGFP-Cl, spermine and sucrose as described above and pelletedand washed as described above. OVCAR-3 cells were grown in 96 wellplates as described above.

All transfection experiments were with OVCAR-3 cells in tissue culture,as described above. The OVB-3 conjugate were added directly to a 40 mMliposome stock and diluted into HBSS without calcium or magnesium. Thefinal total lipid concentration was 4 mM for all samples. 10% heatinactivated fetal bovine serum was added to each sample. 20 gl of a60/40 mM Ca/Mg stock was added to every ml of liposome solution justbefore addition to tissue culture plates. Experiments were performed inquadruplicate.

Liposomes were incubated for transfection of OVCAR-3 cells as describedabove. Total cell number and transfection activity were assessed byfluorescence measurements as described above.

The results are shown in FIG. 13. The fluorescence of EGFP corrected fortotal cell number by the calcein blue reading was plotted againstvarious conditions for transfection. FIG. 13 shows that the conjugatesare always much more active than the corresponding free polylysines interms of transfection, suggesting that the activity is not mediated bythe polylysine portion of the conjugates alone. The data also show thatthe folate conjugates enhance transfection.

EXAMPLE 14

Fusogenic Ahacyl-PE Liposomes: Enhancement of Ex Vivo Binding ofLiposomes to OVCAR-3 Ascites Cells by Polylysine Transferrin

As the first step to testing in vivo transfection, the binding ofliposomes to human OVCAR-3 ascites cells removed from a mouse xenograftwas examined. Liposomes were prepared as described in Example 1. As inExample 1, both DNA-containing and empty liposomes were labeled with arhodamine-PE fluorophore.

A series of experiments was performed using OVCAR-3 cells recovered fromthe ascites fluid of SCID mice. The lavage was accomplished by injectionof 6 ml of PBS into the peritoneal cavity of a mouse that had beeninjected with 1×10⁷ cells 40 days earlier. The recovered cells werepelleted and washed with PBS and resuspended at an estimated density ofapproximately 1-2×10⁶/ml (from hemacytometer) in HBSS without Ca and Mg.Aliquots of 10, 20 or 50 p,l were placed in the round-bottom wells of apolypropylene 96-well plate. HBSS was added to adjust the total volumeof each well to 100 p,l. At this point the plate was spun in acentrifuge, then a fine needle aspirator was used to collect the fluid.After a wash in HBSS, 50 gl of a concentrated (about 10×) cell-freeintraperitoneal fluid collected from OVCAR-3-bearing SCID mice andadjusted with 20 p,l/ml of a 60/40 mM Ca²⁺/Mg²⁺ stock was added to eachwell. Then 50 gl of each liposome sample with added Ca²+/Mg²⁺ was addedto each well, and the wells incubated at 37° C. for 3 hours. Afterincubation, the wells were washed (by centrifugation) 3 times with HBSS(without Ca and Mg) and resuspended in RPMI 1640 with 10% heatinactivated fetal bovine serum and 5 mM sodium butyrate. The cells weremonitored by fluorescence microscopy.

The results are shown in FIG. 14. FIG. 14 reveals that pKT substantiallyenhanced binding of the liposomes to the OVCAR3 cells. There may havebeen some transfection as well. The dramatically enhanced binding seenhere ex vivo would be expected to ultimately greatly enhance in vivotransfection.

EXAMPLE 15

In Vivo Transfection of Intraperitoneal OVCAR-3 Cells byPolylysinetransferrin Targeted Liposomes

In this example we treated a mouse bearing OVCAR3 tumor in a mannersimilar to the model presented in Son,. Cancer Gene Therapy 4, 391;1997, incorporated herein by reference. The treatment involved injectinga very high tumor cell number into the mouse and pretreating withcisplatin before transfection.

Two high cell number OVCAR-3 SCID mice were set up as in Example 6 onday 1. On day 42, 0.2 ml of 0.5 mg/ml cisplatin was injected IP. On day46, each mouse received an IP injection of a liposome preparation asfollows: 0.5 ml of pEGFP-Cl-containing liposomes (NC1₂-DOPE/DOPC, 70/30)were injected IP (one mouse received liposomes with the pZeoLacZ plasmidencapsulated rather than the pEGFP-Cl plasmid). The liposome stock was40 mM total lipid, was diluted 1:1 with 1 mg/ml pKT, and was constitutedin Hanks Balance Salt .Solution (HBSS) without Ca²⁺ or Mg²⁺. Just beforeinjection, 20 p/ml of a stock of 60 mM and 40 mM Ca+² and Mg²⁺,respectively, was added, which brought Ca²⁺ and Mg²⁺ to 1.2 and 0.8 mMrespectively. The same liposome injections followed on days 49 and 52.On day 53, 0.5 ml of the same 20 mM butyrate solution in HBSS withoutCa/Mg was injected IP. On day 54, both mice were sacrified andperitoneal 6 ml PBS lavage was performed on each mouse to obtain ascitescells. A fairly low number of cells was obtained, probably as a resultof the platinum treatment. Some peritoneal wall and mesentery tissue wasalso dissected and frozen for future analysis. The ascites cells werepelleted and washed 2 times with PBS, collected into 0.5 ml and diluted10× for observation and photographs. The pZeoLacZ-treated cells wereresuspended in 5 ml of cold PBS and the pEGFP-treated cells wereresuspended in 10 ml of cold PBS. A 0.3 ml aliquot of each was diuted to1 ml in a plastic cuvette containing PBS and O.D. read at 650 nm againstbuffer blank. The readings were pZeoLacZ-0.836; pEGFP-0.618. A roughestimate of the pEGFP sample was that it contained 1₂e6 cells/ml. ThepZeoLacZ sample was diluted by adding 1.76 ml PBS to approximate thecell concentration of the other sample. Aliquots were taken from thesefor chemiluminescent β-galactosidase assays, and the results are shownin FIG. 15. The EGFP sample serves as a control for the pZeoLacZ.

FIG. 15 shows that significant β-galactosidase activity occurred in thepZeoLacZ transfected cells, but only at the background level in thepEGFP control. The data confirms that in vivo transfection occurred.

EXAMPLE 16

In Vivo Transfection of OVCAR-3 Cells Under Different ConditionsMediated by Polylysine-Transferrin Targeted Liposomes

Previous examples provided evidence for pKT-mediated transfection invivo using a high cell number OVCAR-3 IP system in the SCID mice. Wealso utilized a lower cell number model where more mice could betreated.

11 SCID mice (female CB17 from Taconic) were injected on day 1 with1×10⁷ OVCAR-3 cells from tissue culture. The cells were allowed to growuntil day 73. On day 73 the mice were injected intraperitoneally (IP)with 0.2 ml of 0.5 mg/ml cisplatin in PBS. On day 77, 80 and 83, themice received IP injections of liposomes or lipid complexes. Four typesof DNA-containing systems were injected (see below). On day 84, 0.5 mlof 20 mM sodium butyrate in HBSS w/o Ca/Mg was injected IP. On day 85,peritoneal lavage was performed as in Example 4. Cells from lavagesamples were collected for analysis as described above. Assays forβ-galactosidase were performed with a chemiluminescent substrate as inExample 3

Group 1: Liposomes encapsulating pCMVR plasmid (for β-galactosidaseexpression) at a total lipid concentration of 40 mM were mixed in equalvolume with a stock of 2 mg/ml polylysine-transferrin conjugate (pKT).The liposome mixture received 12 μ per ml of a stock of 60 mM Ca²⁺ and40 mM Mg²⁺ just before injection of 0.8 ml of this mixture into theperitoneal cavity of each of 4 mice.

Group 2: Exactly the same protocol except that liposomes containing thepEGFP-C1 plasmid were used.

Group 3 DC-cholesterol/DOPE (⅔) liposomes were complexed with pCMVβplasmid DNA. A 4mM stock of the above liposomes (prepared by sonicationin a bath sonicator from a rehydrated film) in 20 mM HEPES, pH 7.0, wasdiluted 1:1 in distilled deionized water to give a 2 mM stock. A stockof pCMV(3 plasmid DNA was diluted in water to 1 mg/ml. Equal volumes ofthe stocks were mixed approximately 15 minutes before intraperitonealinjection as described in examples above. 0.3 ml were injected IP intoeach of 2 mice.

Group 4: Exactly the same as the above DC-cholesterol complexes, ,except that pEGFP-Cl plasmid DNA was used. 0.3 ml was injected IP into 1mouse.

The liposomes comprised /V-Cl₂-DOPE/DOPC ( 70/30) prepared sterile andas in the Assays and Methods section, including pelleting and washing.The pKT conjugate is the same as discussed above. The data are shown inFIG. 8.

FIG. 16 reveals that the pKT-mediated liposomal transfection of the theIacZ gene clearly worked in vivo in this IP system.

Though the invention has been described with reference to specificembodiments, those of ordinary skill in the art will recognize thatvarious modifications, omissions, changes and/or substitutions may bemade without departing from the spirit and scope of the inventiondefined in the appended claims.

1-9. (canceled)
 10. A method of introducing a bioactive agent into thecytoplasm of a cell, said method comprising: a) preparing a fusogenicliposome, said fusogenic liposome comprising a lipid bilayerencapsulating a bioactive agent; b) electrostatically linking atargeting moiety to said fusogenic liposome through a linking moiety toform a targeted liposome; c) contacting said targeted liposome with acell; d) dissociating said targeting moiety from at least a portion ofthe lipid bilayer of said targeted liposome to form an exposed lipidbilayer portion; e) interacting said exposed lipid bilayer portion ofsaid targeted liposome with the membrane of said endosome such that saidbioactive agent is released into the cytoplasm of said cell.
 11. Themethod of claim 10, further comprising the steps of electrostaticallylinking a stabilizing moiety to said fusogenic liposome, anddissociating said stabilizing moiety from said at least a portion of thelipid bilayer contemporaneously with said dissociating of said targetingmoiety.
 12. (canceled)
 13. The method of claim 1 1, wherein saidstabilizing moiety is at least one moiety selected from the groupconsisting of polyethylene glycol, polyvinylpyrolidone, a dextran, apolyamino acid, methyl-polyoxazoline, polyglycerol, poly(acryloylmorpholine), and polyacrylamide.
 14. The method of claim 10, wherein thelipid bilayer comprises an N-acylphosphatidylethanolamine (NAPE). 15.The method of claim 10, wherein said targeting moiety is at least onemoiety selected from the group consisting of a vitamin, transferrin, anantibody, sialyl Lewis X antigen, hyaluronic acid, mannose derivatives,glucose derivatives, cell specific lectins, galaptin, galectin,lactosylceramide, a steroid derivative, an RGD sequence, EGF,EGF-binding peptide, urokiase receptor binding peptide, athrombospondin-derived peptide, an albumin derivative and acombinatorial molecule.
 16. The method of claims 10, wherein saidlinking moiety is at least one moiety selected from the group consistingof polylysine, protamine, polyethyleneimine, polyarginine, polyacrylate,a spermine derivative, cytochrome c, an annexin, heparin sulfate, anaminodextran, polyaspartate, polyglutamate, a polysialic acid, andpoly(2-ethylacrylic acid).
 17. The method of claim 10, wherein thebioactive agent is selected from the group consisting of a nucleic acid,an antiviral agent, an antibacterial agent, an antifungal agent, anantimetabolic agent, an antineoplastic agent, a sterol, a carbohydrate,an amino acid, a peptides, a protein, a dye, a radiolabel, a radiopaquecompound, a fluorescent compound, a mydriatic compound, a bronchodilatorand a local anesthetic.
 18. The method of claim 16, wherein thebioactive agent is a condensed nucleic acid.